Phd Thesis Elisa Puzzolo Last

INTERNATIONAL SCHOOL OF ADVANCED STUDIES S.I.S.S.A.-I.S.A.S. Cortico-cerebral development in the gray short-tailed opossum Monodelphis domestica _________ PHD THESIS _________________ Candidate: Elisa Puzzolo Supervisor: Prof. A. Mallamaci 2008-2009 TABLE OF CONTENTS ABSTRACT __________________________________________________________ 4 INTRODUCTION _____________________________________________________ 5 1. Organization and Development of the nervous system _________________ 5 1.1. From neural plate to neural tube _________________________________ 5 1.2. Organization of the adult mammalian cerebral cortex _________________ 8 1.3. Determination of the pallial field _________________________________ 9 1.4. Antero-posterior patterning ____________________________________ 10 1.5. Dorso-ventral patterning ______________________________________ 13 1.6. Specification of cortical area identities ___________________________ 16 1.6.1 Patterning centers involved in cortical arealization ________________ 17 1.6.2 Transcription factors involved in areal identities _________________ 19 2. Mouse Cortical Development _____________________________________ 23 2.1. Cortical Progenitors in Rodents _________________________________ 24 2.2. Neuronal subclass specification _________________________________ 30 2.3. Migration in Rodents _________________________________________ 33 2.3.1 Radial migration __________________________________________ 33 2.3.2 Tangential migration _______________________________________ 36 2.4. Development of thalamo-cortical afferents ________________________ 39 3. Comparative aspects of cortico-cerebral developmen t _________________ 41 3.1. Telencephalic organization in vertebrates _________________________ 41 3.2. Class-specific areal restrictions of neurons with distinct laminar identities 43 3.3. Evolutionary increase in the SVZ _______________________________ 46 3.4. Evolutionary increase in cortical interneurons _____________________ 47 4. Marsupials ____________________________________________________ 49 5. Aims of the thesis _______________________________________________ 51 MATHERIALS AND METHODS _______________________________________ 52 1. Animals _______________________________________________________ 52 2. Bromodeoxyuridine administration ________________________________ 52 3. Organotypic cultures ____________________________________________ 52 4. Brains Electroporation __________________________________________ 53 5. Histology ______________________________________________________ 53 6. Immunofluorescence ____________________________________________ 54 6.1. Immunofluorescence protocol __________________________________ 54 6.2. Antibodies used _____________________________________________ 54 7. In situ hybridization protocol _____________________________________ 55 7.1. Preparation of Dig-labelled RNA probe __________________________ 55 7.2. Pretreatment of sections _______________________________________ 56 7.3. Hybridization and washing of sections ___________________________ 56 7.4. Digoxigenin revelation _______________________________________ 57 8. Imaging and confocal microscopy _________________________________ 57 9. Sample sizing __________________________________________________ 57 RESULTS ___________________________________________________________ 58 1. Molecular diversification of neurons belonging to different neocortical laminae and their radial migration ____________________________________ 58 2. Does a Basal Progenitors compartment exist in the opossum? __________ 65 3. The apical proliferative compartment and its dynamics _______________ 70 4. Distribution and generation of cortical GABAergic cells ______________ 72 5. Post-neuronal histogenesis: generation of astrocytes and oligodendrocytes 74 DISCUSSION ________________________________________________________ 76 CONCLUSIONS _____________________________________________________ 78 SUPPLEMENTARY MATERIAL ______________________________________ 80 REFERENCES _______________________________________________________ 83 ACKNOWLEDGMENTS ______________________________________________ 94 ABSTRACT The marsupial South-American short-tailed opossum, Monodelphis domestica, is an appealing animal model for developmental studies on cortico-cerebral development, since the opossum cortex mainly develops after birth and newborns are particularly suitable for early ex-utero micro-surgical manipulations of this structure and the entire CNS. Opossum have been also largely employed as an ideal substrate for regenerative studies, since the pup is able to regenerate connections between neurons of the cerebral cortex and spinal cord upon experimental trauma. Moreover, branching from the common mammal ancestor about 180 My ago, Marsupials might provide a valuable tool for tracing evolutionary origins of key traits peculiar to the eutherian central nervous system (CNS). Until recently, the cortico-cerebral marsupial development has been prevalently investigated by methods of classical histology, but several features of Monodelphis corticogenesis were still unknown. By taking advantage of molecular tools set up for developmental studies in Placentals and availability of Monodelphis domestica genomic sequence, we tried to fill gaps in our knowledge of opossum corticogenesis, studying in particular: origin of cortical neurons, their laminar differentiation and their migration profiles, from their birthplaces to their final layer positions. We found many similarities between marsupial and placental corticogenesis, as for neuron generation, their laminar diversification and “inside-out” migration. This allowed us to establish a comparative time-table of mouse and opossum corticogenesis. One major difference emerged from our study. In the opossum, projection neurons are mainly born from apical progenitors and a basal proliferative compartment is hardly detectable. 4 Introduction INTRODUCTION 1. ORGANIZATION AND DEVELOPMENT OF THE NERVOUS SYSTEM The development of the nervous system is a complex process that begins during early embryogenesis with the induction of neural cells, then the formation of the neural plate and afterwards the establishment of the primordial nervous system of the embryo. The vertebrate nervous system has two anatomical distinct parts: the central nervous system (CNS) which consists of the brain and the spinal cord and the peripheral nervous system (PNS), which is composed of the sensory organs and of the autonomic and the enteric nervous system. 1.1. From neural plate to neural tube The central nervous system (CNS) arises during early development from the neural plate, a cytologically homogeneous sheet of neuroepithelial cells (NE) ( Fig. 1 ). The neural plate is induced from the underlying mesoderm . The neuroepithelial cells are thought to acquire distinct properties depending on the positions within the CNS primordium to yield enormously divergent neuronal cell types at specific locations. The neural plate is subdivided into molecularly distinct domains with characteristic locations. Figure 1 | Electro-scanning of E9.5 mouse embryo showing the lumen and the neuroepithelium of the neural tube . (B) Magnification of boxed area in (A). The green asterisk identifies a mitotic cell at the apical surface. Images modified from www.med.unc.edu 5 Introduction During neurulation, the neural plate folds up at the margins and forms the neural tube (Fig. 2). Figure 2 | Schematic view of the neural tube formation and the signaling sources involved . 1) opened neural plate; 2) neural grove formation; 3) closed the neural tube; 4) delaminating neural crests. Image modified from Nicholls et al. , 4 th edition, 2001. Before the closure of the neural tube, the neural plate becomes subdivided along the anteroposterior axis , into three distinct domains, corresponding to the three primary vesicles: the prosencephalon (the forebrain), the mesencephalon (midbrain), and the rhombencephalon (the hindbrain) (Fig. 3A). These initial regions become further subdivided as development proceeds : the prosencephalon will give rise to diencephalon and telencephalon; the rhombencephalon to metencephalon and mylencephalon ( Fig. 3B). Figure 3 | Schematic view of the anterior neural tube . (A) three-vesicle stage; (B) five-vesicle stage. Image adapted from Nicholls et al. , 4 th edition, 2001. 6 Introduction The telencephalic vesicles occupy the most rostral position of the neural tube and can be subdivided into a dorsal (pallial) and a ventral (subpallial) territory. The ventral telencephalon or subpallium is further subdivided into two main domains, called basal ganglia: the more ventrally located is the medial ganglionic eminence (MGE), precursors of the globus pallidum, the more dorsal is the lateral ganglionic eminence (LGE), which generates the striatum. A third eminence called caudal ganglionic eminence (CGE) supplies for the amygdala (Fig. 4 ). Figure 4 | (A) Schematic view of a coronal section through the developing mouse telencephalic vesicle at E12. (B) Sagittal view of the embryonic vertebrate telencephalon as a transparent structure to reveal the ganglionic eminences CGE, caudal ganglionic eminence; CTX, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; OB, olfactory bulb. Images modified from Molyneaux et al. , 2007 (A) and Corbin et al. , 2001 (B). The cerebral cortex originates from the dorsal pallium. It includes four components, termed medial, dorsal, lateral and ventral pallium and corresponding to the hippocampus, the neocortex, the

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