Thesis
Reference
Postnatal positioning of neuronal precursors in the dorso-medial limbic cortex : a potential involvement of Wnt signaling pathways
ZGRAGGEN, Eloisa
Abstract
Les couches corticales du cerveau des mammifères sont générées par des événements précis de migration neuronale; l'élucidation des mécanismes qui gouvernent ceux-ci est d'une importance capitale pour comprendre comment leur dysfonctionnement pourrait contribuer aux désordres neurodéveloppementaux. Dans la première partie de cette étude nous avons investigué les événements de migration corticale à partir de la zone ventriculaire/sous-ventriculaire dans les rats nouveau-nés. Nous avons découvert la présence d'une population de précurseurs glutamatergiques migrant dans le cortex limbique dorsomédial (CxLdm) pendant la première semaine postnatale. Dans le cortex granulaire retrosplénial ces cellules forment une population neuronale bien définie de la couche II. Dans la deuxième partie de ce travail nous avons testé l'hypothèse que la voie de signalisation Wnt joue un rôle dans le positionnement corticale de ces neurones. Nos données préliminaires démontrent, pour la première fois, que la voie Wnt est requise pour la migration des derniers neurones pyramidaux du CxLdm.
ZGRAGGEN, Eloisa. Postnatal positioning of neuronal precursors in the dorso-medial limbic cortex : a potential involvement of Wnt signaling pathways. Thèse de doctorat : Univ. Genève et Lausanne, 2012, no. Neur. 93
URN : urn:nbn:ch:unige-238485
DOI : 10.13097/archive-ouverte/unige:23848
Available at:
http://archive-ouverte.unige.ch/unige:23848
Disclaimer: layout of this document may differ from the published version.
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FACULTÉ DES SCIENCES
DOCTORAT EN NEUROSCIENCES des Universités de Genève
et de Lausanne
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Professeur Jozsef Z. Kiss, directeur de thèse
Postnatal Positioning of Neuronal Precursors In the Dorso-Medial Limbic Cortex:
A Potential Involvement of Wnt Signaling Pathways
THÈSE
Présentée à la Faculté des Sciences de l’Université de Genève
pour obtenir le grade de Docteure en Neurosciences
par
Eloisa ZGRAGGEN
de
Lugano (Tessin)
Thèse N°93 Genève
Imprimeur: Uni Print Center de Genève 2012
Remerciements
Je veux tout d’abord remercier les membres du jury de ma thèse: le professeur Jozsef Z. Kiss, le professeur Denis Jabaudon, le professeur Jean-Pierre Hornung et le professeur Dominique Muller.
Je suis particulièrement reconnaissante au professeur Jozsef Z. Kiss du fait qu’il m’a donné la possibilité de travailler dans son laboratoire, pour sa supervision, pour son encouragement et pour son constant enseignement.
Je tiens à remercier toutes les personnes qui ont collaboré à ce projet, qui ont partagé avec moi la fascinante et complexe vie de la recherche et qui m’ont motivée et aidée pendant des innombrables occasions: en particulier Michiko Kanemitsu, Gaël Potter, Michael Boitard, Inge Roman, Béatrice King, Cynthia Saadi, Sylvie Chiliate et Vladimir Petrenko, avec les anciens et les nouveaux membres des laboratoires du professeur Jozsef Z. Kiss, du professeur Alexandre Dayer et du docteur Laszlo Vutskits.
Ma gratitude va enfin à ma famille et à tous mes amis, en premier Antonio Romeo, qui ont partagé avec moi les surprises et les efforts de cette longue aventure et qui m’ont assuré un inoubliable soutien en renouvelant toujours en moi la curiosité et le désire de connaître.
Un remerciement spécial va à mon mari Giuseppe, son amour à incroyablement illuminé cette dernière période de travail intense.
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TABLE OF CONTENTS
1 ABSTRACTS ... 4
1.1 Résumé en français ... 4
1.2 Abstract ... 6
2 INTRODUCTION ... 8
2.1 Overview of the mammalian cerebral cortex ... 8
2.2 The dorso-medial limbic cortex (dm LCx) ... 13
2.2.1 Anatomy ... 13
2.2.2 Functions ... 15
2.3 Layer positioning of cortical projection neurons during development ... 18
2.3.1 Overview of cortical development ... 18
2.3.2 Radial migration of cortical projection neurons ... 22
2.4 Importance of neuronal positioning in neonatal pathologies ... 26
2.5 Regulation of radial migration ... 30
2.5.1 Extrinsic factors ... 30
2.5.2 Intracellular mediators ... 37
2.6 Wnt signaling in neocortical development ... 41
2.6.1 Overview and general functions ... 41
2.6.2 Involvement in neocortical development ... 44
2.6.3 Potential roles in cortical cell positioning ... 46
2.7 Goal of the thesis ... 49
3 RESULTS AND DISCUSSIONS 1
STPART: ... 51
3.1 Results ... 51
3.1.1 Postnatal radial migration into the dorso-medial limbic cortex (dm LCx) ... 51
3.1.2 A pre-migratory pyramidal precursor pool in the neonatal dm VZ/SVZ ... 54
3.1.3 Postnatally migrating cells towards the dm LCx cortex display a pyramidal phenotype and position in layer II ... 59
3.1.4 Postnatally migrating cells are generated at embryonic ages ... 61
3.1.5 Dynamics of cell migration into the dm LCx ... 62
3.1.6 Postnatally migrating cells differentiate into dendritic bundles (DB) forming cells in the retrosplenial granular cortex (RSGC) ... 65
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3.2 Discussions ... 69
3.2.1 Origin of postnatally migrating cells ... 70
3.2.2 Postnatally migrating cells form a homogenous pyramidal cell population in LII of the dm LCx ... 71
3.2.3 Radial migration in the early postnatal period ... 73
3.2.4 The final fate of postnatally migrating pyramidal precursors ... 75
3.2.5 Conclusion ... 76
4 RESULTS AND DISCUSSIONS 2
NDPART: ... 77
4.1 Results ... 77
4.1.1 Mapping canonical Wnt activity in dm LCx ... 77
4.1.2 Migrating LII-directed pyramidal cells respond to Wnt/β-catenin signaling ... 80
4.1.3 Loss/gain of function of Wnt signaling in LII-directed cells of dm LCx ... 81
4.1.3.1 A loss of function approach ... 82
4.1.3.1.1 Validation of RNAiDvl constructs... 83
4.1.3.1.2 Knock-down of Dvls does not affect migration of LII-directed pyramidal cells ... 85
4.1.3.2 A gain of function approach ... 87
4.1.3.2.1 Overexpression of Dvl2 mimics activation of canonical Wnt signaling in vitro ... 87
4.1.3.2.2 Dvl2-overexpression affects cell positioning of LII-directed pyramidal cells ... 89
4.1.3.2.3 Dvl2 overexpression decreases migratory speed of LII-directed pyramidal cells ... 94
4.1.3.2.4 Dvl2 overexpression promotes process formation in LII-directed cells ... 96
4.1.3.3 Pilot experiment with beta-catenin gain of function ... 100
4.2 Discussions ... 102
4.2.1 Wnt/β-catenin signaling activity in migrating LII-directed cells of dm LCx ... 102
4.2.2 Studying Wnt signaling by loss/gain of functions of Dvl ... 105
4.2.2.1 Dvl genes silencing does not impact migration of LII-directed cells of dm LCx ... 106
4.2.2.2 Wnt-Dvl2 gain of function experiments ... 108
4.3 Conclusion and perspectives ... 114
5 MATERIALS AND METHODS ... 120
5.1 Animals ... 120
5.2 Nomenclature ... 120
5.3 In vivo lentivector injections ... 121
5.4 Design and production of lentivectors ... 121
5.5 In utero vectors injection and electroporation ... 122
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5.6 Plasmids construction... 123
5.7 Cell culture ... 125
5.8 Cell transfection and Luciferase Reporter Assay ... 125
5.9 Tissue processing and immunohistochemistry... 126
5.10 X-gal staining ... 128
5.11 Birthdating experiments ... 128
5.12 Cortical slice preparation and time-lapse imaging ... 129
5.13 Image and data analysis ... 130
6 REFERENCES ... 134
Abbreviations ... 152
List of Figures ... 154
List of Publications ... 156
Acknowledgments……… 157
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1 ABSTRACTS
1.1 Résumé en français
Les couches corticales sont générées par des événements précis et coordonnés de migration cellulaire, et le cortex des mammifères est particulièrement vulnérable aux troubles de la migration. L’élucidation des mécanismes et de la régulation de ces événements migratoires est d’une importance capitale pour comprendre comment les altérations de ce processus pourraient contribuer aux désordres neurodéveloppementaux. Les deux composants majeurs du cortex limbique dorsomedial, cortex cingulaire et cortex rétrosplenial, jouent un rôle fondamental permettant la connexion entre la formation hippocampale et le complexe amygdalien avec les régions néocorticales; de plus, ils ont été impliqués dans un éventail de fonctions cognitives tel que l’émotion, l’attention, et la mémoire spatiale. Bien que l’on ait beaucoup appris concernant la structure et les fonctions de ces deux régions corticales, leurs développement reste peu connu.
Dans la première partie de cette étude, nous avons investigué les événements de migration dirigée vers le cortex à partir de la zone ventriculaire/sous-ventriculaire (ZV/ZSV). Dans ce but, nous avons injecté dans la ZV/ZSV des vecteurs lentiviraux codant pour des protéines fluorescentes. Nous avons découvert la présence d’une population de précurseurs glutamatergiques postmitotiques, localisée à l’angle dorsomedial de la ZSV: ces cellules entrent en migration et se positionnent dans le cortex limbique dorsomédial pendant la première semaine postnatale. Cette migration est robuste et guidée par la glie radiale. L‘observation de la migration de ces cellules pyramidales par videomicroscopie (time-lapse) a révélé des comportements et morphologies remarquablement différents entre la zone intermediaire (substance blanche) et la matière grise corticale. Dans le cortex granulaire retrosplénial, la grande majorité de ces cellules se développe en une population neuronale bien définie: occupant la couche II, et se caractérisant par l’expression du marqueur Satb2 et la formation de faisceaux de dendrites dans la couche I.
Ces résultats apportent de nouveaux éléments concernant le processus migratoire de la dernière
5 population de cellules pyramidales prenant place dans le cortex limbique dorsomédial. De plus, ils offrent l’opportunité d’étudier les mécanismes moléculaires et d’identifier les facteurs de régulation impliqués dans le positionnement cortical d’une population cellulaire précise. De telles investigations sont d’une importance capitale pour déterminer la contribution de certaines mutations génétiques, ainsi que de facteurs environnementaux négatifs, dans le développement d’une lamination corticale anormale donnant lieu à des désordres neurodéveloppementaux.
Dans la deuxième partie de ce travail, nous avons testé l’hypothèse que la voie de signalisation Wnt puisse jouer un rôle dans la séquence d’étapes amenant au positionnement des neurones pyramidaux de la couche II du cortex limbique dorsomédial. On sait depuis longtemps que cette voie de signalisation joue un rôle crucial dans plusieurs aspects de la corticogenèse, dont la prolifération des progéniteurs neuronaux et la différentiation neuronale; cependant, son implication directe dans la régulation de la migration cellulaire ainsi que dans le positionnement cortical n’a pas encore été étudié. Nous avons développé différents outils moléculaires que nous avons ensuite introduits dans les précurseurs pyramidaux de la ZV/ZSV au moyen d’injections intraventriculaires et d’electroporation in utero. Ainsi nous avons, dans un premier temps, mis en évidence que la voie de signalisation Wnt -catenin est active dans les cellules pyramidales en migration. Puis, notre stratégie basée sur un gain/perte de fonction a révélé des effets remarquables sur les cellules en migration vers la couche II; en particulier des altérations de la polarité, de la locomotion et du positionnement.
En conclusion, ces données préliminaires démontrent, pour la première fois, que la voie de signalisation Wnt est requise pour la migration radiale de la dernière population de cellules pyramidales du cortex limbique dorsomédial.
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1.2 Abstract
Cortical layers are generated by precisely coordinated cell migration events and the mammalian cortex is particularly susceptible to disorders of migration. Elucidating the mechanisms and regulation of migratory events is therefore crucial to understand how alterations in this process might contribute to neurodevelopmental disorders. The two major components of the dorso-medial limbic cortex (dm LCx), the cingulate (CGC) and retrosplenial (RSC) cortices occupy a critical position in linking the hippocampal formation and the amygdaloid complex to neocortical areas and have been implicated in a range of cognitive functions such as emotion, attention, and spatial memory. While much has been learned about the adult structure and functions of these cortical regions, little is known about their development.
In the first part of this study, we investigated cortically directed migration of neurons from the ventricular/subventricular zones (VZ/SVZ) by injecting lentiviral constructs coding for fluorescent proteins directly into the SVZ. We discovered a prominent pool of postmitotic cortical glutamatergic precursors localized in the dorso-medial corner of the SVZ. Cohorts of precursor cells exited from this premigratory pool and migrated into the dm LCx during the first postnatal week. This migration was robust and guided by the radial glia scaffold. Time-lapse imaging demonstrated that this population of postnatally migrating pyramidal cells displays important morphological and behavioral changes as cells traverse the intermediate zone (white matter) and subsequently the cortical gray matter. In the granular retrosplenial cortex, the large majority of these cells give rise to a specific cell type: Satb2+, layer II pyramidal cells with typical dendritic bundles in layer I. Overall these findings provide the first insight into the migratory pattern and dynamics of late-generated neurons settling into the dm LCx. Moreover they offer the opportunity to study the molecular mechanisms and identify regulatory factors involved in cell positioning of a defined cell population; such investigations are of crucial importance to predict the contribution of genetic mutations and adverse environmental factors on aberrant cortical layering underlying neurodevelopmental disorders.
7 The hypothesis we tested in the second part of this study is that Wnt signaling pathways may play a role in the sequential steps leading to the postnatal positioning of LII pyramidal neurons of dm LCx. It has long been known that this signaling pathway plays a central role in many aspects of corticogenesis, including neural progenitor proliferation and neuronal differentiation, but its direct role in regulating cell migration and positioning remained unknown.
We have developed a set of molecular tools and introduced them into proliferative cells of VZ/SVZ using in utero, intraventricular injections and electroporation. We found that the Wnt/β- catenin signaling is active in radially migrating cells. Moreover, the inspection of migrating cells following variable survival times revealed remarkable effects of gain-of-function strategies, including alteration in cell polarity, cell locomotion and positioning. Together these preliminary findings provide the first demonstration that Wnt signaling is required for the radial migration of late-generated pyramidal precursors of dm LCx.
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2 INTRODUCTION
2.1 Overview of the mammalian cerebral cortex
The cerebral cortex first appears in lower vertebrates when the most anterior part of the neural tube, the telencephalon, gives rise to a flat homogenous sheet of neural tissue named “pallium”.
Through evolution the pallium increases in size and complexity, covering the outer space of the cerebrum; it results as the major component of the dorsal telencephalon in mammals and becomes the seat of cognitive functions and the most important evolutionary acquisition in these species (Fig. 1) (Kriegstein 2006; Clowry 2010).
Figure 1: Genesis and evolution of the cerebral cortex. A) Schematic drawings illustrating pallial formation from the telencephalon which grows until enwrapping the diencephalon. B) (Top) Representative brain sections of rodents, carnivores and primates (form the Comparative Mammalian brain collections at http://brainmuseum.org/), illustrating the relative increase in cortical convolution; (Bottom) Sections across of the mouse, macaque monkey and human brains drawn at approximately the same scale; note the large difference in surface of about 1: 100: 1000 X in mouse, macaque monkey and human, respectively.
The pallial growth reaches an impressive level in complex mammals, and particularly the most external pallial part, so that the pallium becomes convoluted, i.e. being composed of gyri (folds) and sulci (interfolds). The name of mammalian pallium, i.e. “cerebral cortex”, reminds to this feature. Furthermore, the highest pallial expansion is found in humans, with an incredible
9 surface area of 1820 cm2, a volume of 490 cm3 and an average thickness of 2.7 cm (Ramakers 2005; Pierani and Wassef 2009). These values confers to the human cerebral cortex unique features in term of anatomical complexity, driving the emergency of intellectual abilities and making of the cerebral cortex the structure that more distinguishes humans from the other species (Lui 2011).
The mammalian cerebral cortex is firstly organized in the horizontal plane, being composed by superimposed neuronal layers (Fig. 2) (Molyneaux 2007). The more evolved part, called neocortex, is composed of 6 layers numerated from one to six starting from the pial surface. In mammals, this cortex represents 90% of the cortical volume whereas the remaining part is occupied by allocortex, i.e. “different” cortices, which are more ancient and involve 3 layers only (Pierani and Wassef 2009; Lui 2011). Cortical neurons are further arranged in adjacent vertical microcircuits (named columns) that involve all layers and confer to the cortex a radial structure (Fig. 2) (Jones and Rakic 2010).
Figure 2: Structural features of the neocortex. Schematic diagram of a mouse brain coronal hemisection showing the localization of neocortex with respect to allocortex; the cellular structure of a representative portion of the neocortex (red boxed area) is illustrated on the right by a picture of a Nissl-stained coronal section; note the radial and horizontal arrangement of cortical neurons.
Another characteristic of the the mammalian cortical sheet is its heterogeneity; indeed regional cortical differences were first described by looking at cytoarchitectonical criteria, such as the thickness of each layer and the predominant cell type, and gave rise to the first cortical
10 map, also known as Brodmann map (Brodmann 1909; von Economo and Koskinas, 1925). Later on, regional differences of the cerebral cortex were mapped according to the type of function they support resulting in a specie-specific functional map whose areas do not necessarily correlate with those in Brodmann’s map (Nauta & Feirtag 1986).
Figure 3: Cortical arealization in the embryonic mouse brain. A-D) Schematic drawings of the embryonic brain from a rostro-lateral view (A), of a brain hemi-section (B) and whole-section (C-D) from a dorsal view; they show:
A) Patterning centers localization, B) Gradient of expression of early working transcription factors (TFs), C) Embryonic field formation by collaborative activity of TFs and morphogenetic proteins, D) Map of mture functional areas. Abbreviations: CTX, cortex; LGE, MGE, CGE, lateral, medial and caudal ganglionic eminence respectively;
OB, olfactory bulb; M1, S1, V1, A1 motor, somatosensory, visual, auditory primary cortex respectively. (Images adapted from Hansen et al. 2011).
Cortical areas are commonly organized in “primary” and “association” cortices: the primary cortices comprise: 1) the area sending the primary motor input, i.e. the primary motor cortex, and 2) the areas receiving primary sensory inputs of a unique single modality, i.e. visual, auditory and somatosensory primary cortices; all the other cortices are called “associative” as they receive
11 and process second order information of one or multiple modalities. Thus, each cortical area distinguishes from the others by the projections it receives and by the target of its own efferent projections. In particular, a distinct set of thalamic projections innervate each cortical area (Nauta & Feirtag 1986). The growth of such projections into the developing cortical areas is known to play a modulatory role in the process of area specification (Clowry 2010); on the other hand the principal regulators of thalamic fiber ingrowth are specific morphogenetic proteins which are secreted by restricted scattered regions of the embryonic cortical primordium called patterning centers (Fig. 3). These morphogenic proteins are distributed in concentration gradients and function as transcriptional regulators; their patterns of activity superpose with the differential expression pattern of early working transcription factors (TFs). The results is a zone-specific
“cocktail” of such arealizing elements representing the first traces of the cortical areas and giving rise to a “protomap” or “embryonic field” (Rakic 1988). According to the current hypothesis, the cell growth, position and fate is regulated differently in each neuroepithelial portion by the combinatorial activity of TFs, which hence specifies the size and identity of future neuronal cortical populations (Fig. 3) (Dehay and Kennedy 2007; Mallamaci 2011).
The cerebral cortical layers are populated by multiples neuronal subtypes; we distinguish two main functional classes: the glutamatergic projections neurons, or pyramidal neurons, that represent 80% of the total and send excitatory inputs to internal and external circuits, and the GABA-ergic interneurons corresponding to 20% of the total neuronal population and acting at the level of the local circuit. Different subtypes of projection neurons and interneurons are further specified through the activity of subtypes-specific pool of TFs as mentioned above and they mainly differ by morphology, birthdating, molecular identity, and axonal target (Fig. 4) (Molyneaux 2007; Vitalis and Rossier 2011).
Indeed, each cortical layer mainly distinguishes from the other by the target of the residing projection neurons: subcortical projecting neurons (corticofugal) are settled in layers V and VI;
in layer II, III and IV corticortical projection neurons are located, including neurons having
12 callosal projections to the contralateral cortex (Fig. 4) (Fame 2011b). Upper layer neurons (II/III) are evolutionarily younger and their number increase in complex species, being a major factor of cortical size’ enlargement. As a consequence, cortico-cortical projections represent an evolutionary acquisition and drive the development of cognition; as a matter of fact, in humans more than 90% of cortical connexions are cortico-cortical (Rakic 2009).
Figure 4: Neuronal composition of the neocortex. A) (Left) Drawing of a coronal adult brain section illustrating the proportion of pyramidal projection neurons (black) and interneurons (red) within the cortex (boxed region).
(Right) Schematic representation of different cortical interneurons subtypes (red) and their interaction with projection neurons (black). B) Simplified scheme depicting laminar organization of the different projection neurons subtypes according to the axonal projections. Note that a small contingent of callosal-projecting neurons (gray) resides also in layer 5. Main subtype-specific combinations of TFs are listed on the left. (Figures adapted from Batista-Brito and Fishell 2009, and Gaspard and Vanderhaeghen 2011).
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2.2 The dorso-medial limbic cortex (dm LCx)
2.2.1 AnatomyA particular gyrus is found in the medial aspect of the primate brain, lying just above the corpus callosum and embracing it; as a matter of fact, it is called “cingulate”, meaning “like a belt”. The cortex occupying the cingulate gyrus and continuing in the cingulate sulcus is thus named “cingulate cortex”. In lower mammals, whose brain is not convoluted, the cingulate gyrus occupies the medial wall of cerebral hemispheres from the corpus callosum to the dorsal brain surface (Fig. 5) (Vogt 2009 Ch. 1; Vogt, Ch. 22, Paxinos, 2004).
Figure 5: Comparison of primate and rodent cingulate cortex. A) Photographs of monkey and rat medial brains at the same scale, showing morphology of respective cingulate areas. In the monkey, cingulate and callosal sulci were separated to expose the hidden cortical areas (24 c,c’,d and 29/30 respectively). (B) Topographic maps of human and rat cingulate cortex (flattened reconstructions from a medial brain view). Main cortical regions are marked by colors. (Images modified from Vogt 2009 and from Vogt et al., Ch.2, Paxinos 2004).
Although it forms a single and continuous structure, the cingulate cortex is structurally and functionally heterogeneous, including at least twenty subregions in humans. An important aspect is its “transitional” identity, meaning that, instead of being a proper isocortex, it presents a series of cytoarchitectural variations among its subregions; thus, it appears like a bridge between the
14 one- and six-layered adjacent areas, the indusium griseum/subiculum and the parieto-occipital association areas respectively (Vogt 1995; Vogt 2009, Ch.3). In addition, unlike the other cortices which have a transverse gyrification, the cingulate gyrus is directed parasagittally.
Because of its transitional nature and parasagittal position, Broca (1878) classified the cingulate cortex in the “limbic lobe”, i.e. the lobe containing all structures with “bestial” features (like the hippocampal formation, the parahippocampal gyrus and the infralimbic part).
Consequently, the cingulate cortex is also named the “dorso-medial limbic cortex” (dm LCx) (Morgane and Mokler 2006). Over time, discordance emerged among neuroanatomists about the classification of cingulate cortex as part of the limbic lobe, about its parcellation and about the identification of subregions in different species (Vogt Ch.22, Paxinos 2004; Vann 2009). The criteria that emerged as the most useful to discriminate between the multitude of functional areas in the brain and to recognize correspondent cortices among different species (such as humans and rats) was introduced by Brodmann (1909). He numbered the cortical areas according to cytoarchitectural features instead of lobe subdivision. Furthermore, a huge work of comparative cytoarchitectural analyses, and, more recently, studies based on electrical stimulation and functional imaging, led to an agreement on a modified Brodmann cortical map, which allows direct comparison among species (Vogt, Ch.22, Paxinos 2004; Vann 2009). According to this new view, the primate dm LCx is divided in four main regions (Fig. 5). The first two compose the anterior cingulate cortex (ACC, areas 24, 32, 33) which is defined on the basis of absence of the internal granular layer (layer IV); the perigenual ACC (pACC, a. 25, 32, 24a/b) is located anteriorly and the midcingulate cortex posteriorly (MCC, a. 24a’, 24b’). MCC is followed by the third and fourth regions, called posterior cingulate cortex (PCC, a. 23, 31) and retrosplenial cortex (RSC) respectively. The latter is composed by the retrosplenial granular cortex (RSGC, a.
29a/b), by the retrosplenial dysgranular cortex (RSDC, a. 30), and by the ectosplenial cortex (a.
26).
15 Although many similarities are found between human and rodent dm LCx, the dm LCx of rodents is not convoluted, implying that some areas present in humans are missing in rodents (a.
23, 26, 31, 24a’b’) and only three main regions are identified: pACC (a. 25, 32, 24a/b), MCC (a.
24’) and RSC (a. 29, 30) (Fig. 5). Indeed, it is still not possible to define an exact equivalence between cingulate cortical areas of rodents and primates, and in the actual model the correspondence is based on structural similarities only. For instance, in rats and humans the Rg (a. 29) is identified by the presence of a poorly differentiated layer 4, but this region does not have the same position in the two species and differences also exist at the level of connections and cytology (Morris 2000; Vann 2009; Vogt, Ch.22, Paxinos 2004).
While there are still ongoing studies on improving the identification and classification of functional units in primate and lower mammal’s dm LCx, there is general agreement that the existing similarities between rat and humans dm LCx are enough to consider the rat as a useful specie to model human diseases involving this cortex and particularly the RSC (Vogt and Peters 1981; Vogt, Ch.22, Paxinos 2004).
2.2.2 Functions
First of all, the dm LCx was recognized to take part in the the circuit of emotions (such as the Papez circuit) based on its reciprocal connections with the amygdale, the anterior thalamic nuclei, the hypothalamus and neocortical areas (Vogt, Ch.22 in Paxinos 2004; Morgane and Mokler 2006). It was proposed to play a role in transferring the impact of emotions on cognitive and autonomic functions.
The advances of last decades in axonal tracing techniques as well as in functional neuroimaging significantly improved our understanding of this hidden brain region, particularly in its role of visuomotor and sensorymotor integration and in memory-related tasks. Although there is an intense connection between the anterior and the posterior part of cingulate cortex,
16 major functional differences have been identified between these two parts and between their subregions (Vann 2009; Vogt, Ch.22, Paxinos 2004).
As a first example the ACC, and in particular area 24b, has a main role in visuomotor integration and nociceptive processing; area 24b contains nociceptive neurons and corticospinal motor neurons, it is connected with secondary motor areas, and it is provided with visual stimuli by area 24b’ through visual cortices or RSC mediation. The role of area 24b seems to be the generation of motor activity in response to visual stimuli, such as the head turning following a visual cue, and motor and autonomic reflexes (such as cardio-vascular) following peripheral pain (Vogt 2009, Ch.14). This area not only mediates motor reactions to direct sensory stimuli but also memorizes the rewarding properties of a visually-guided motor behavior so that the next reaction would be influenced by this experience; in this way it seems to play a role in motivation control.
As a second example the RSC constitutes a seat of elaboration of multiple sensory inputs since it has the merit of converging, through direct and indirect pathways, multiple sensory information concerning smell, pain, auditory and visual inputs and position perception (Fig. 6) (Vann 2009). In addition, the RSC has dense reciprocal connexions with the anterodorsal and anteromedial thalamic nuclei and with the hippocampus (Wyss and Van Groen 1992b; Vogt 2009, Ch.13), thus providing a pathway from the thalamus to the hippocampic areas. These two properties allow the RSC to play a particular role in focusing attention to relevant emotional events, and in associating memories to sensory inputs. As a consequence, the RSC develops its major task in a memory-related cognitive function, i.e. spatial navigation (Kravitz 2011). Animal lesion studies have actually revealed that the activity of this cortex is critical to create a spatial representation, to code the spatial position of the body and to recall known routes (Fig. 6) (Vogt, Ch.22, Paxinos 2004). fMRI studies on human subjects demonstrated the collaboration of RSC with the PCC in the recall of episodic information, thus confirming the role of RSC in the memory process in primates. Moreover, lesions touching this brain area are proven to impair
17 memory and can provoke anterograde amnesia including topokinetic disorientation (Valenstein 1987; Rudge and Warrington 1991; Takahashi 1997). Finally, some of the most common human neurological disorders affecting memory, such as Alzheimer disease, present a compromised RSC (Vann 2009).
Figure 6: Integrative role of the human retrosplenial cortex (RSC). Illustration indicating that distinct types of information are converged from different brain regions and elaborated together in the RSC. The integrative role of RSC underlies the emergence of cognitive functions such as episodic memory and spatial navigation. Abbreviations:
ATN, anterior thalamic nuclei. (Figure adapted from Vann et al. 2009)
In summary, based on the widespread intracortical connexions of this area, the role of the dm LCx appears much more than just a simple mediator of emotions; it is indeed involved in various functional tasks, some of which being rather complex and underlying major cognitive aspects (Vann 2009). Researchers are now working to better characterize the functions of the dm LCx and to identify the mental processes which require its activity. It is also under evaluation whether specific tasks characterize the different subregions of the RSC, as suggested by their distinct pattern of connections (Sugar 2011). Animal models aimed to distinctively analyze and
18 functionally test particular brain subregions could significantly contribute in answering to the latter question. Researchers from the group of Wyss are providing good examples in this direction (Wyss and Van Groen 1992a; Van Groen 2004).
It is finally to notice that the dm LCx has been implicated in major psychiatric pathologies, such as schizophrenia (Bluhm 2009), that are associated with developmental disturbances (Vann 2009). In line with this observation, neurons of its regions appear particularly vulnerable to perinatal adverse effects of stress (Rivarola and Suarez 2009) and hypoxia (Li 1998). However, while the adult structure, connectivity and functions of this crucial cortex have been characterized or are on the process of being revealed, few studies have indeed provided information on its development (Bayer 1990a, b; Barbe and Levitt 1991; Ichinohe 2003; Miro- Bernie 2006; Miyashita 2010; Fame 2011b).
2.3 Layer positioning of cortical projection neurons during development
2.3.1 Overview of cortical developmentAlthough projection neurons and interneurons form together the cortical circuits, they invade the cortex independently and arise in different proliferative regions (Fig. 7A). Whereas projection neurons are generated in the dorsal pallium and migrate radially to invade the above lying developing cortex, interneurons are generated in the subpallium and migrate tangentially to the dorsal pallium (Marin 2010); from there they invade the cortical plate (CP) by switching to a multimodal mode of migration, termed “oblique migration” (Nadarajah 2003; Yokota 2007). In higher mammals a large proportion of interneuron also arise in the dorsal proliferative regions (Jones 2009; Petanjek 2009); however, proliferative and migratory events of these cells are distinct from those of projection neurons.
19 The delicate process of production and positioning of cortical projection neurons occurs in multiple phases and by means of multiple progenitor cells (Fig. 7B).
Figure 7: Origin of cortical projection neurons. A) Diagram of a coronal embryonic brain section illustrating the origin and migratory route of excitatory cortical neurons (black) and of cortical interneurons (red). B) Schematic drawing of dorsal germinative layers, depicting evolution along time and diversity of projection neurons progenitor cells. Abbreviations: LV, lateral ventricle; VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate; MZ, marginal zone; NE, neuroepithelial cell; IPC, intermediate progenitor cell; RGC, radial glia cell; SNP, short neural precursor; oRGC, outer RGC. (Image modified from Dehay and Kennedy 2007).
At the beginning of corticogenesis the dorsal neuroepithelium, also called ventricular zone (VZ) (Boulder Committee 1970), is composed of neuroepithelial (NE) cells only. These are highly polarized cells, with an apical process attached to the lumen of the ventricle, and a basal radial process fixed to the pial surface. NE cells divide rapidly and symmetrically to increase the germinal pool and gradually acquire astrocytic characteristics to become the so-called “radial glial cells” (RGC) (Götz and Huttner 2005; Bystron 2008).
In a second phase (E10.5), NE/RG cells start to divide asymmetrically, giving rise to postmitotic neuroblasts or rapidly proliferating intermediate progenitors (IPs) which resemble to RG but have a shorter basal process not contacting the pia; these IPs are named short apical neural precursors (SNPs). At this time, the VZ reaches its maximal size and is mainly devoted to the production of lower layer neurons. In a third phase, NE/RG cells give rise to another sort of intermediate progenitors (IPs), also called basal progenitors (BPs) (Bystron 2008; Merot 2009);
these cells loose both the apical endfeet and the ascending process and they divide in the basal part of the VZ. With the progression of corticogenesis the accumulated BPs form a second
20 germinal layer, the so-called subventricular zone (SVZ) (Boulder Committee 1970), which is mainly devoted to the production of upper layers neurons. In primates (Smart 2002; Hansen 2010) and, to a lesser extent, in rodents (Shitamukai 2011; Wang 2011), the SVZ is enriched by an additional population of neuronal progenitors constituted by the outer RG (oRG) cells, i.e. RG cells which lose contact with the ventricle and migrate in the outer part of SVZ (oSVZ).
Currently, oRG are considered as the main cause of the amplified number of neurons underlying the expansion and convolution of primate corticogenesis (Lui 2011).
In initial stages of cortical development, the earliest produced postmitotic neurons are the Cajal-Retzius cells (Götz and Huttner 2005), that migrate and accumulate above the SVZ forming the first cortical compartment, i.e. the preplate. After that, there is a sequential production of the different neuronal populations which settle in layers II-VI. This process follows the rule of an inside-out gradient: i.e. lower layers neurons are generated, migrate, and settle in the cortical plate before upper layers neurons, which thus have to migrate through older cells to find their final position (Fig. 8). Firstly, layer VI neurons settle in the middle of the preplate (E12), breaking this compartment in two transitory regions: the upper preplate or marginal zone (MZ), located close to the pial surface, and the deeper preplate or subplate, located close to the germinal layers. Layer VI neurons keep adjacent to the subplate remaining in the lowest part of the cortical plate so that the following neuronal population will have to cross layer VI neurons. Being the last formed, layer II neurons migrate through all the previously positioned layers to gain their position close to the MZ, which, in turn, becomes the layer I.
During this process, another transitory compartment, the intermediate zone (IZ), forms just above the germinal layers blending with the subplate; this region is traversed by growing axons and migrating cells and, towards the end of the migratory period, it turns into the white matter (WM) compartment (Bystron 2008).
After the generation of layer II pyramidal neurons, which corresponds to the end of gestation in rodents, the production of pyramidal precursors is ceased and the the SVZ starts to shrink;
21 precursor cells give rise to glial cells only whereas RG cells perform their final division generating glia, ependymal cells, or postnatal stem cells. The VZ/SVZ are replaced by the ependymal layer and the subependymal zone (SEZ), the latter producing olfactory bulb interneurons during the whole postnatal life (Bonfanti and Peretto 2007; Bystron 2008).
Figure 8: The process of cortical lamination. A) Schematic drawings depicting (Top) how later born neurons migrate past earlier born neurons, and (Bottom) how cortical lamination progresses by the “inside-out” way. B) Different classes of projection neurons are born in overlapping temporal waves. Abbreviations are as in previous figure and: E, embryonic day; IZ, intermediate zone; WM, white matter; PP, preplate; SP, subplate; SEZ, subependymal zone. Romanic numbers indicate layers. (Images modified from Molyneaux et al. 2007).
Two main subsequent processes follow the settling of neurons within the cortex and underlie the formation of the cortical network: the neuronal differentiation and the formation of functional connections between neurons. Basically, postmigratory neurons first transform morphologically by increasing the cell body size, by extending the axon toward the target, and by developing a huge dendritic arbor; then they establish synaptic contacts and progressively acquire mature electrical and neurochemical features. A critical period follows, where early formed synapses are strengthened or eliminated (synaptic pruning), and similarly, neurons which are not properly connected undergo cell death. All these events are thought to be driven and modulated by the interplay of genetic program, cells interactions, and specific patterns of early spontaneous and experience-dependent electrical activity (Ramakers 2005; Hanganu-Opatz 2010).
22 Before concluding this section, it is worth spending a few words about the regulation of these processes. The whole development of cortical neurons, from neuronal production to neuronal specification and differentiation, is mainly governed by a genetic program which depends on the temporal and spatial interplay of multiple basic-helix-loop-helix (bHLH) TFs. Neuronal fate commitment particularly involves the activator proneural bHLH TFs (Mattar 2008), and the repressor-type bHLH Hes genes; the latter maintain progenitor cells in an undifferentiated proliferative state or favor the gliogenic fate depending on the developmental cell state (Kageyama 2008). Proneural bHLH TFs also promote neuronal migration (Heng 2008), whereas the balance between various proneural bHLH TFs governs the choice of glutamatergic versus GABAergic phenotype, and participates in the specification of a defined neuronal subtype (Merot 2009; Rubenstein 2011). It is important to notice that, although cell-intrinsic genetic- programs collaborate with extrinsic factors in guiding laminar fate specification, cell transplantations experiments indicate that the fate potentialities of cortical neuronal precursors is progressively restricted with the advancement of corticogenesis, likely as a consequence of epigenetic mechanisms which are under cell cycle control (Kageyama 2008).
Since my thesis is focused on the mechanisms of regulation of projection neurons positioning within the cortex, in the following section I describe the cortical radial migration in detail.
2.3.2 Radial migration of cortical projection neurons
The appropriate position of cortical neurons is established by highly regulated sequential migratory events making the mammalian cerebral cortex particularly vulnerable to disorders of cell migration (Marin 2010; Govek 2011). According to the traditional view, pyramidal excitatory neurons generated in the VZ/SVZ by radial glia or by IPs, migrate radially away by following radial glial processes. However, recent investigations, and in particular video time- lapse studies (Nadarajah 2001, 2003; Tabata and Nakajima 2003; Hatanaka 2004; Noctor 2004;
23 Loturco and Bai 2006; Tabata 2009), revealed that radial migration is a complex multistep process as follows:
Figure 9: Schematic representation of the multistep migratory process of cortical projection neurons.
The red dot inside the cells indicates localization of the centrosome. Abbreviations are as in previous figures.
a) First (step 1, Fig. 9), newly generated postmitotic neurons pass through a phase of slow migration in the SVZ/IZ where they dynamically extend and retract multiple processes while moving randomly. One hypothesis is that this so called “multipolar phase of migration” has a significant role for the axonal sprouting; the precursor of the axon seems actually to be selected at this moment among the many processes of the multipolar cell.
b) The second step (step 2, Fig. 9) of the radial migratory journey consists in cell re-polarization and in the acquirement of a bipolar shape: the cell selects a trailing process, which becomes oriented toward the back, and a leading process which is normally the most prominent and extends on the direction of migration; these two processes are the precursor of the axon and of the apical dendrite respectively. The leading process progressively associates with a basal radial glial fiber, thus acquiring stability and providing tension on the actin cytoskeleton inside the cell.
24 At that moment cells are positioned in the lower IZ and are ready to invade the cortical plate (Kriegstein and Noctor 2004; Stiess and Bradke 2011).
Figure 10: The locomotion process on radial glial fibers. A) Schematic of the migratory cycle of a locomoting neuron. Alterations in neuronal morphology occur through changes in the cytoskeleton. B) Schematic drawing of representative structures in a radially migrating neuron. Note that actin and motor proteins are enriched at the rear part of the nucleus and at the proximal aspect of the leading process. (Images modified from Valiente and Marin 2010).
c) Next, the true step of radial migration into the cortical plate occurs by locomotion on a leading substrate, the radial glial scaffold (step 3, Fig. 9); as illustrated in Figure 10A this mode of migration is characterized by two phases of somal translocation. In a first phase, the leading process grows in length and establishes new adhesion complexes with the glial fiber; thanks to the traction forces thereby generated, many organelles are pulled forward into a cytoplasmic dilatation formed on its proximal part; among them are the Golgi and the Centrosome which thus
25 become oriented toward the CP. In a second phase, the nucleus moves into the cytoplasmic swelling: the nucleus is linked to the centrosome through a cage of microtubules and it is pulled forward thanks to the work of microtubules-associated dyneins and of surrounding actomyosins (Fig. 10B). The end of the migratory cycle occurs with the rupture of the adhesions at the back of the cell and with the concomitant retraction of the trailing process. The migratory cycle restarts when a new extension from the leading process occurs. Hence, thanks to the radial glial structure, young pyramidal neurons proceed straightforward to the MZ and display a typical saltatory pattern due to the repeated nuclear jumping into the cytoplasmic swellings. Because many cells migrate at the same time and on the same glial fiber, radial columns of migrating cells develop from the IZ to the outer border of the CP.
d) Once the leading process of a migrating cell reaches the MZ, it anchors to the pial surface forcing the cell to a new migratory mode, called translocation (step 4, Fig. 9) (Nadarajah 2001;
2003); this movement is created by the progressive shortening of the leading process, pulling forward the cell body (Miyata 2001). This phase, called “terminal translocation”, allows the correct positioning of cells close to the MZ and is a required step for the proper formation of a new neuronal layer.
It is important to note that radial cell migration has been typically studied on dorso-lateral regions of the neocortical primordium, leaving unexplored from this point of view the dm region, i.e. the dm LCx. The few available studies on dm LCx development provide information on the general pattern of neurogenesis or focus on dendritic and axonal growth (Bayer 1990a, b; Barbe and Levitt 1991; Ichinohe 2003; Miro-Bernie 2006; Miyashita 2010; Fame 2011b). Neurogenetic studies indicate that, similarly to neocortex, the dm limbic region develops in an ‘‘inside-out’’
fashion but in terms of timing it differs from adjacent somatic cortices (Bayer 1990a,b): in a given layer of neocortex, neurons of medial regions are generated later than those located in more lateral subdivision, while, on the contrary, in the dm LCx more medial parts contain the older neurons. Moreover, since the dm LCx is located in a parasagittal-directed gyrus,
26 progenitors of this cortex have to migrate on curved paths to join the medial MZ, thus differing from neuronal progenitors of lateral cortex which migrate on vertical paths. These elements suggest that the pyramidal migratory process in the dm LCx may not follow the same pattern and dynamics as in the adjacent somatic neocortex; new studies are thus needed to improve our knowledge on dm LCx development. Such investigations might also contribute to reveal whether alterations of dm LCx development are implicated in the pathogenesis of schizophrenia; indeed, such neuropsychiatric disorder is known from both human studies and animal models to be related to dysfunctions of dm LCx (Bluhm 2009). In fact, as I explain in the following section, abnormal cortical layering is a major cause of brain pathologies.
2.4 Importance of neuronal positioning in neonatal pathologies
Recent advances in genetics of humans pathologies affecting the CNS, have led to the discovery that many of them take origin from cortical malformation related to neuronal migration disorders during development. In parallel, new studies evaluating the interplay between cognitive deficits, failure in information processing and early developmental malformations, have found that impaired cortical lamination is a major cause of such cognitive deficits (Meng 2005; Threlkeld 2007).
Proper formation of cortical structure is a requirement for normal cognitive functions; if neurons are misplaced, the structure is not stable and problems occur like in a domino effect. In fact, aberrant neuronal positioning provokes defects in the connection among cortical neurons, modifying the wiring with other brain regions and thus affecting cognition (Paul 2007).
Therefore, the phase of cortical layering is a high vulnerable period during development. As outlined above, a successful cortical layering requires the coordination of several stages: timed neurogenesis, departure of neuroblasts from the VZ, migration to the cortical plate, settling in the appropriate layer, and neuronal maturation. Indeed, the last step is necessary to consolidate the
27 cortical layering before the establishment of neuronal connections. Disruptions at any of these stages result in malformations of cortical development (MCD), even leading to a large long-term impact on processing information. Mental retardation and epilepsy in children are classical precocious effects of MCD; after such diagnoses, neuroimaging analyses now rapidly allow to recognize cortical malformation. Also, MCD can be at the origin of dyslexia, specific syndromes such as Miller–Dieker and Tourette’s, and psychiatric illnesses such as schizophrenia and autism (table 1) (Mcmanus and Golden 2005; Guerrini and Parrini 2010).
The pathogenesis of MCD is known to be triggered by environmental factors that can be physical (e.g., ionizing radiation, ultrasonic waves, and heat), chemical (e.g., drugs and alcohol), and biological (e.g., neurotropic viruses); on the other hand recent analyses revealed that genetic mutations also play a significant role in generating MCD. The type and seve rity of the cortical dysmorphy (i.e. malformation) are indeed determined by both the degree of
28 insult/mutation and the developmental stage at which this insult occurs (or at which the effects of mutations display) (Pang 2008).
Detecting, studying and modeling MCD of genetic origin may be critical to recognize the origin of neuropsychiatric disturbances of unknown pathologies. Recent research focused on the use of animal models allowing to provoke loss-of-function of genes involved in neuronal migration and to observe its effect on the cortical structure (Spalice 2009; Guerrini and Parrini 2010; Liu 2011). These studies have largely enhanced our knowledge of the effects of a single gene mutation in terms of migratory defects and cortical dysmorphies, thus contributing to a better description and classification of MCD (Barkovich 2001, 2005; Francis 2006; Pang 2008).
As proposed by Pang et al. (2008) (Table 1), MCD can first be grouped according to the defective stage of cortical development; further criteria of classification are the resulting cortical dysmorphy, the causative gene (if any) and the functional impairment (i.e. the clinical aspects).
A first group of cortical dysmorphies concerns MCD issued by defects in neuronal proliferation.
Besides decreasing the number of neurons, defects in neurogenesis, such as premature cell cycle exit, induce rapid differentiation of neural progenitor cells and result in an inappropriate migration to ectopic locations (Mitsuhashi and Takahashi 2009). Typical examples of brain malformations issued from abnormal neurogenesis are microcephaly (“small brain”) and focal cortical dysplasia, indeed one of most severe cause of epilepsy in children.
A second group of MCD consists of cortical dysmorphies which result from a direct defect in the neuronal migratory process, i.e. heterotopia and lissencephaly of type I (Spalice 2009).
Heterotopia, meaning “different localization”, is a cortical malformation characterized by a cluster of neurons in an abnormal location; depending on the location, heterotopias can be classified as periventricular nodular, subcortical or marginal glioneuronal. Lissencephaly means
“smooth brain” and refers to the paucity of normal gyri and sulci; the severity of this type of malformation can vary from agyria or pachygyria to a normal gyral pattern with subcortical band heterotopias (Ozmen 2000). In type I (or classical) lyssencephaly many neurons fail to migrate to
29 the cortical plate so that there are only four layers instead of six. By disrupting formation of leading process, or by inhibiting the coupling of nucleus-centrosome coupling during nucleokinesis, centrosomal defects are a well-known source of migratory failure and thus of classical lissencephalic phenotype (Fig. 11A,B).
Figure 11: Migratory defects generate cortical malformations. A) Schematic drawings of abnormal cortical layering in distinct neuronal migration disorders and relative MRI (a,b,c) or T2-weighted (d) images of patients’
brains. B) Schematic drawings illustrating examples of migratory defects and associate cortical malformations.
Abbreviations: SCZ, subcortical zone; VZ, ventricular zone; WM, white matter. (Panels adapted from: Spalice et al.
2009; Liu 2011; Manzini and Walsh 2011).
A second pathological subtype of lyssencephaly, named cobblestone (or type II), is classified in a third group of MCD since it results from a missed arrest during cell migration. Such defect in the migratory arrest phase implies that neurons overmigrate and accumulate outside the cortex forming ectopias; hence neuronal layers are disorganized and the cortex has a nodular appearance. Principal causes of cobblestone are defects in basal lamina assembly and disruptions in radial glial orientation (Fig. 11A,B).
30 Finally, a fourth type of MCD are cortical dysmorphies issued by impairments of neuronal maturation or connection formation, whose proper functioning is required for a correct cortical layering. A first example is polymicrogyria, which appears when the cortex develops with an excessive number of smaller gyri and thus enlarged sulci. Another example is schizencephaly, meaning “cleft brain”, since in this malformation the cerebral hemispheres are crossed by a cleft which connects extra-axial subaracnoid spaces and ventricles (Pang 2008).
Thanks to animal models reproducing cortical malformations, to the progress in neuroimaging allowing direct observation of the migratory behavior, and to the combination of the neuroimaging techniques with molecular biology and genetic approaches, the knowledge of the molecular machinery and regulatory elements involved in radial cortical migration has incredibly improved. Indeed, many regulatory elements have been first recognized by analyzing mutated genes in human patients with brains syndromes (Ross and Walsh 2001); on the other hand, investigations on the signaling pathways involving these elements are greatly helping in understanding the causality of a number of neurodevelopmental malformations and diseases.
Surely, there is a great expectation that the resulting progress will lead to excogitate new therapies aimed at reducing the pathological effects on the cortical development due to a lost or mutated gene function.
In the next chapter I have summarized our knowledge on the regulatory elements involved in radial migration.
2.5 Regulation of radial migration
2.5.1 Extrinsic factorsAlthough the intrinsic genetic program and the locomotion capability of migrating cells underlie the row progression of the migratory process, research over several decades has
31 revealed that the correct running of each step needs to be tightly controlled by multiple extrinsic factors; they can act by means of very different mechanisms like triggering the cell motility, functioning as guidance cues or mediating neuronal adhesion on radial glial processes (Ayala 2007; Marin 2010). In the following, I review the main molecules involved in regulation of radial migration, and when known, the mechanisms which underlie their regulatory role (an illustrative summary is provided in Figure 12). I have grouped these factors in two classes according to their identity: substrate-anchored factors and diffusible messengers.
a) Substrate-anchored factors
The substrate-anchored factors are membrane-bound cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins. The reciprocal hemophilic or heterophilic recognition of these proteins underlies adhesive or repulsive interaction of a cell with the extracellular environment; a specific set of these molecules indeed characterizes distinct migratory phases and can differently influence cell movement; their main activity in regulation of radial neuronal migration seems to be the choice of the substrate for migration and directing the extension of the leading process (Franco and Muller 2011). As a matter of fact, impaired activity of CAMs/ECM proteins generally causes a detachment of migrating cells from RGCs and/or reduces the length of the leading process thus decreasing the rate of migration.
A well-known ECM molecule involved in pyramidal cell positioning, is laminin. When laminin is depleted in migrating cells, the extension of the leading process is actually retarded and the glial-neuron interaction is disrupted, resulting in cell migration defects. The mechanisms underlying this effect are still under investigation. During cortical development, laminin seems to be secreted by migrating neurons and deposited along the radial glial fibers; the molecules which mediate interaction of laminin with migrating neurons seem to be CAMs from the Integrins family. According to the proposed model, laminin would stick on integrins inserted on the neuronal surface and activate downstream pathways regulating the cytoskeleton and the
32 integrin trafficking. As a consequence, neurite would extend favoring the progression of the nucleus (Schmid and Anton 2003).
Although there is no agreement on their precise role and involved isoforms, many evidences indicate that CAMs of Integrin family are implicated in regulation of radial cell migration (Schmidt and Anton 2003; Marin 2010). Integrins are heterodimers containing two distinct chains, the α (alpha) and β (beta) subunits; β1 isoform is expressed ubiquitously in the developing cerebral cortex and is present both on migrating neurons and radial glial cells;
instead, alfa subunits are expressed in a region- and time-dependent way, and the identity of alfa subunits collaborating with β1 in radial migration is not clear. 3 appears the best candidate since it is proven to function as coreceptor for the ECM molecule Reelin (Dulabon 2000) and their interaction may induce the detachment of neurons from radial glia at the MZ (Luque 2004);
moreover, some reports affirm that β1/ 3 integrin is a critical mediator of neuronal adhesion on radial glia, likely by associating with laminin or fibronectin present on the surface of glial processes (Anton 1999; Schmid 2004). In contrast with these studies, Belvindrah et al. (2007) show that β1 integrin is not required for cortical lamination but is instead crucial for glial and neuronal differentiation. Besides indicating that the mechanisms underlying the involvement of integrin in radial migration have still to be clarified, this result underlines the fact that the adhesive properties of a cell are critically involved not only in cell migration but also in regulating the process of cell differentiation. Because precocious cell differentiation can block cell migration, by regulating cell differentiation, integrins may indirectly play a role in cell migration.
A further indirect way by which integrins may influence radial cell migration is through the development of the basal lamina. As a matter of fact ECM components of the basal lamina have been found to act as stop signals for migrating neurons and gaps in basal lamina composition (due to the absence of one or more of these molecules) have suggested to be the cause of neuronal overmigration out of the developing brain (Schmid and Anton 2003; Belvindrah 2007).
33 Moreover, ECM molecules allow the anchorage of radial glial fibers to the meningeal basement membrane and this fact is crucial for the correct maintenance of radial glial cell morphology.
Another family of CAMs, which is worth to be mentioned for its regulatory activity in radial migration, is the family of connexins (Valiente and Marin 2010). The connexins of two juxtaposed cells form intercellular adhesion structures, called Gap junctions, characterized by the presence of a channel allowing cell communication. Specific isoforms of connexins, Cx26 and Cx43, are indeed expressed at the contact point between RGCs and pyramidal migrating neurons during cortical development, and their presence has been reported to mediate neuronal adhesion on radial glial processes. While the channel properties are not involved in this activity (Elias 2007), the intracellular portion of Cx43 seems critical to anchor the cytoskeleton to the adhesive contacts, thus enabling stabilization of the leading process along radial glial fibers and the subsequent translocation of the nucleus (Cina 2009).
Last but not the least, the ECM molecule Reelin (Reln) plays a crucial role for cortical development. Indeed in this context, it seems to act more like a diffusible signaling molecule rather than a substrate-anchored factor: Reln is actually secreted by Cajal–Retzius neurons located in the MZ, it diffuses in the extracellular matrix creating a cortical gradient, and sticks on different transmembrane lipoprotein receptors located on the surface of migrating pyramidal cells. As evidenced by the phenotype of Reln-deficient mice (Caviness and Sidman 1973), Reln occupies a main place in regulation of cortical lamination: in these animals the cortical inside-out gradient of migration is indeed abolished and laminar malformations are found in multiple brain structures causing a lyssencephalic syndrome (Liu 2011). Multiple studies focusing on the activity of Reln have revealed that this molecule can have different impact on migrating cells depending on the contextual situation and on the receptor (Cooper 2008).
First, Reln acts as chemoattractant guidance molecule by increasing growth cone motility and extension, and by increasing filopodia formation (Leemhuis 2010); this role as “enabling cue”
