The human brain, as much as it is “unaccountable” in the eyes of Virginia Woolf, is a marvel. It is the evolutionary increase in brain size, especially in the cerebral cortex, that both allowed Mrs Woolf to create and us to perceive the beautiful imagery that exists in her fictional world. The evolutionary increase in brain size in part reflects the increase in the number of neurons generated during neocortical development. This in turn reflects two principal features of cortical expansion, namely, an increase in the number of neural stem and progenitor cells (from here on referred to as progenitor cells) and their neurogenic potential. Strikingly, in order to cater for this increase in progenitor cells and neurogenic potential, there is a significant expansion and diversification of basal progenitors in the subventricular zone (SVZ). Basal progenitors can be divided into three types: basal intermediate progenitors (bIPs), basal radial glias (bRGs) and transit-amplifying progenitors (TAPs). bIPs are the most abundant progenitors in the mouse SVZ. These cells are non-polar and are Pax6 and Sox2 negative, but Tbr2 positive. They have limited proliferative capacity as they can divide only once to produce two neurons. bRGs and TAPs, on the other hand, are able to undergo multiple rounds of division and exist in higher abundance in gyrencephalic brains (for bRG, in humans up to 50% versus mouse 5% at mid-neurogenesis). The morphology of bRGs are reported to be dynamic (fluctuating between states of having process(es) to none), whereas TAPs are generally described to be non-polar during mitosis. bRGs are known to express Pax6 and Sox2 but not Tbr2 while TAPs are known to express both Pax6 and Tbr2. The increase in the proportion of these self-renewing basal progenitors (more specifically bRGs) might allow for cortical expansion. Hence, the main objective of this doctoral work was to generate more bRGs in the mouse dorsal telencephalon, the region that ultimately develops to become the cerebral cortex. To achieve this objective, two approaches were used– (i) a general approach by microinjecting a pool of ferret poly-A+ RNA and (ii) a candidate approach by conditionally expressing the transcription factor Pax6. In the general approach, the microinjection technique was first established and validated in an organotypic slice culture of the mouse dorsal telencephalon. A pool of ferret poly-A¬+ RNA extracted at P1, the developmental stage corresponding to the peak of bRG production, was then microinjected into the dorsal telencephalon. We hypothesized that at the peak of bRG production, the “instructive” messages on how to generate bRG would be at their peak. Hence, by introducing these “instructive” messages into a apical radial glia (aRG), these cells would thus “know” how to generate bRGs. At 24 h after microinjection, only aRGs, the predominant progenitor residing in the ventricular zone during mid-neurogenesis were recovered. At 48 h after microinjection, however, 75% of cells that translated the ferret poly-A¬+ RNA had a morphology reminiscent of bRG. These cells were located away from the ventricular surface and had a basal but not apical process. We conclude from these experiments that we did indeed generate bRG-like cells in the mouse dorsal telencephalon via microinjection of the ferret poly-A¬+ RNA. In the candidate approach, this work aimed to conditionally express Pax6, a transcription factor that has been linked to proliferation and neurogenesis in aRG. More specifically, as there is a significant increase in the number of Pax6 positive cells (bRGs) in the SVZ of gyrencephalic animals during mid-neurogenesis, we wanted to recapitulate this phenomenon in the mouse dorsal telencephalon, where Pax6 is normally downregulated. To achieve this, the Tis21–CreERT2 mouse was used. Tis21 is a pan-neurogenic marker that is switched on once aRG switches from a proliferative division (i.e. 1 aRG⇒2aRG) to a neurogenic division (i.e. 1aRG⇒1aRG+1bIP). Consequently, the neurogenic aRGs and its progeny, bIPs would thus be Tis21 positive. By conditionally expressing Pax6 in Tis21 positive aRGs, the ectopic expression of Pax6 was successfully induced in the SVZ of the mouse dorsal telencephalon. Interestingly, conditional expression of Pax6 increased the percentage of proliferating cells in the SVZ. However, instead of producing more bIPs as predicted by the neurogenic division of Tis21 positive aRGs, these cells had the cell morphology, transcription factor expression profile, and division-type of bRGs and/or TAPs. Thus, using the conditional expression of Pax6 we were able to generate more bRG-like progenitors in the mouse dorsal telencephalon. The fate of these conditionally expressing Pax6 progenitors at a later stage was then investigated. A phenotypic change in the behaviour of neurons generated was observed. Instead of migrating into the cortical plate, cells that were highly expressing Pax6 formed a heterotopia at the SVZ or intermediate zone, suggestive of Pax6 interfering with neuronal migration. Interestingly, of those lowly expressing Pax6 cells that successfully migrated to the CP, a disproportionate majority became upper layer neurons. As the fate of neurons are dependent on their date of birth (i.e early born neurons are normally found in the deep layer while late born neurons are normally found in the upper layer), the increase in the upper layer neurons is consistent with the fact that conditionally expressing Pax6 delayed the birth of these neurons by delaying neurogenesis in order to increase the number of proliferative divisions. Interestingly, this increase in upper layer neurons is consistent with the difference between small- and large-brained species. In conclusion, through this work more bRGs was successfully generated in the mouse dorsal telencephalon through two distinct but complementary approaches.

The human brain, as much as it is “unaccountable” in the eyes of Virginia Woolf, is a marvel. It is the evolutionary increase in brain size, especially in the cerebral cortex, that both allowed Mrs Woolf to create and us to perceive the beautiful imagery that exists in her fictional world. The evolutionary increase in brain size in part reflects the increase in the number of neurons generated during neocortical development. This in turn reflects two principal features of cortical expansion, namely, an increase in the number of neural stem and progenitor cells (from here on referred to as progenitor cells) and their neurogenic potential. Strikingly, in order to cater for this increase in progenitor cells and neurogenic potential, there is a significant expansion and diversification of basal progenitors in the subventricular zone (SVZ). Basal progenitors can be divided into three types: basal intermediate progenitors (bIPs), basal radial glias (bRGs) and transit-amplifying progenitors (TAPs). bIPs are the most abundant progenitors in the mouse SVZ. These cells are non-polar and are Pax6 and Sox2 negative, but Tbr2 positive. They have limited proliferative capacity as they can divide only once to produce two neurons. bRGs and TAPs, on the other hand, are able to undergo multiple rounds of division and exist in higher abundance in gyrencephalic brains (for bRG, in humans up to 50% versus mouse 5% at mid-neurogenesis). The morphology of bRGs are reported to be dynamic (fluctuating between states of having process(es) to none), whereas TAPs are generally described to be non-polar during mitosis. bRGs are known to express Pax6 and Sox2 but not Tbr2 while TAPs are known to express both Pax6 and Tbr2. The increase in the proportion of these self-renewing basal progenitors (more specifically bRGs) might allow for cortical expansion. Hence, the main objective of this doctoral work was to generate more bRGs in the mouse dorsal telencephalon, the region that ultimately develops to become the cerebral cortex. To achieve this objective, two approaches were used– (i) a general approach by microinjecting a pool of ferret poly-A+ RNA and (ii) a candidate approach by conditionally expressing the transcription factor Pax6. In the general approach, the microinjection technique was first established and validated in an organotypic slice culture of the mouse dorsal telencephalon. A pool of ferret poly-A¬+ RNA extracted at P1, the developmental stage corresponding to the peak of bRG production, was then microinjected into the dorsal telencephalon. We hypothesized that at the peak of bRG production, the “instructive” messages on how to generate bRG would be at their peak. Hence, by introducing these “instructive” messages into a apical radial glia (aRG), these cells would thus “know” how to generate bRGs. At 24 h after microinjection, only aRGs, the predominant progenitor residing in the ventricular zone during mid-neurogenesis were recovered. At 48 h after microinjection, however, 75% of cells that translated the ferret poly-A¬+ RNA had a morphology reminiscent of bRG. These cells were located away from the ventricular surface and had a basal but not apical process. We conclude from these experiments that we did indeed generate bRG-like cells in the mouse dorsal telencephalon via microinjection of the ferret poly-A¬+ RNA. In the candidate approach, this work aimed to conditionally express Pax6, a transcription factor that has been linked to proliferation and neurogenesis in aRG. More specifically, as there is a significant increase in the number of Pax6 positive cells (bRGs) in the SVZ of gyrencephalic animals during mid-neurogenesis, we wanted to recapitulate this phenomenon in the mouse dorsal telencephalon, where Pax6 is normally downregulated. To achieve this, the Tis21–CreERT2 mouse was used. Tis21 is a pan-neurogenic marker that is switched on once aRG switches from a proliferative division (i.e. 1 aRG⇒2aRG) to a neurogenic division (i.e. 1aRG⇒1aRG+1bIP). Consequently, the neurogenic aRGs and its progeny, bIPs would thus be Tis21 positive. By conditionally expressing Pax6 in Tis21 positive aRGs, the ectopic expression of Pax6 was successfully induced in the SVZ of the mouse dorsal telencephalon. Interestingly, conditional expression of Pax6 increased the percentage of proliferating cells in the SVZ. However, instead of producing more bIPs as predicted by the neurogenic division of Tis21 positive aRGs, these cells had the cell morphology, transcription factor expression profile, and division-type of bRGs and/or TAPs. Thus, using the conditional expression of Pax6 we were able to generate more bRG-like progenitors in the mouse dorsal telencephalon. The fate of these conditionally expressing Pax6 progenitors at a later stage was then investigated. A phenotypic change in the behaviour of neurons generated was observed. Instead of migrating into the cortical plate, cells that were highly expressing Pax6 formed a heterotopia at the SVZ or intermediate zone, suggestive of Pax6 interfering with neuronal migration. Interestingly, of those lowly expressing Pax6 cells that successfully migrated to the CP, a disproportionate majority became upper layer neurons. As the fate of neurons are dependent on their date of birth (i.e early born neurons are normally found in the deep layer while late born neurons are normally found in the upper layer), the increase in the upper layer neurons is consistent with the fact that conditionally expressing Pax6 delayed the birth of these neurons by delaying neurogenesis in order to increase the number of proliferative divisions. Interestingly, this increase in upper layer neurons is consistent with the difference between small- and large-brained species. In conclusion, through this work more bRGs was successfully generated in the mouse dorsal telencephalon through two distinct but complementary approaches.

Die vorliegende Studie untersucht die Entwicklung des Nervensystems von drei Arten der Höheren Krebse (Malacostraca): Die Leuchtgarnele (Euphausiacea) Meganyctiphanes norvegica und die beiden Zehnfußkrebse (Decapoda) Penaeus monodon (Dendrobranchiata) und Procambarus fallax f. virginalis (Astacida). Auf Basis von Antikörper- und Fluoreszenzfärbungen in Verbindung mit Konfokaler Laser-Scanning Mikroskopie und 3D Rekonstruktionen, umfasst die Studie den Beginn der Axogenese und zeichnet die Entstehung eines axonalen Grundgerüstes in einer umfassenden Abfolge durch die Embryonal- wie auch die Postembryonalentwicklung nach. Die Daten zeigen, dass die drei untersuchten Arten ein allgemeines Muster bei der Entwicklung des Nervensystems teilen. Mittels eines vergleichenden Zuganges wird das gefundene Muster in Hinblick auf die segmentale Körperorganisation der Tiere diskutiert. Insbesondere die Entwicklung des peripheren und des enteralen Nervensystems spielen eine Schlüsselrolle im Prozess der Führung des grundlegenden axonalen Grundgerüstes. Der Vergleich zeigt, dass die medulla terminalis, welche sich bei den Naupliuslarven von M. norvegica und P. monodon in enger Verbindung zu einem Paar sensorischer Frontalorgane entwickelt, eine separate ontogenetische Einheit darstellt, die keinen Teil des dreiteiligen Gehirns der Tiere repräsentiert. Auf Grundlage der phylogenetischen Verwandtschaftsbeziehungen wird die Frage einer möglichen Homologie zwischen diesen sensorischen Organen und den “Frontalfilamenten“ bei nicht-malakostrakten Krebsen, sowie eine neue Interpretation des sogenannten “lateralen Protocerebrums” im Grundmuster der Crustacea-Entwicklung diskutiert. Darüber hinaus liefert die Studie die Identifikation der einzelnen Strukturen des sich entwickelnden stomatogastrischen Nervensystems und enthält eine Zusammenfassung der bisherigen diesbezüglich verwendeten Nomenklatur. Abschließend wird die Hypothese der Entwicklung des Nervensystems als Ergebnis der koordinierten Interaktion dreier unabhängiger Nervensysteme, namentlich dem Zentral-, dem Enteral- und dem peripheren Nervensystem, entwickelt. Die Entwicklung des axonalen Grundgerüstes, als grundlegendes Netzwerk afferenter und efferenter Neuronen für die Verbindungen zwischen diesen drei Systemen, erscheint daher entkoppelt vom Prozess der Segmentierung. / The present study addresses the development of the nervous system in three malacostracans species: the euphausiacean Meganyctiphanes norvegica, and the two decapods Penaeus monodon (Dendrobranchiata) and Procambarus fallax f. virginalis (Astacida). Based on the use of antibody stainings and fluorescent dyes in combination with CLSM and 3D reconstruction, the observations cover the onset of axogenesis and follow the establishment of the axonal scaffold in a consistent and comprehensive sequence through the embryonic and the post-embryonic development. The development of the nervous system reveals a general developmental pattern shared by the three investigated species. With a comparative approach, the observed pattern is discussed with respect to the segmental organization of the animals’ body. In particular, the development of the peripheral and of the enteric nervous systems plays a crucial role in the process of guiding the main axonal scaffold. In this context, the medulla terminalis, which in the nauplius larvae of M. norvegica and P. monodon develops strictly associated to a pair of frontal sensory organs, is proposed as a separate unit and not part of the tripartite brain. The homology of these sensory organs with the “frontal filaments” of non-malacostracan crustaceans and a new interpretation of the so called “lateral protocerebrum” in the developmental ground pattern of the Crustacea are discussed against the current phylogenetic background. Moreover, the present study offers a precise identification of the single structures forming the stomatogastric nervous system and provides a review of the former nomenclature. The interpretation of the labrum as a non-segmental appendage associated to the stomatogastric nervous system is advanced. Finally, the present study proposes the development of the nervous system as the result of the coordinated interaction of three independent nervous systems, i.e. the central, the enteric and the peripheral. As a consequence, the development of the axonal scaffold, i.e. the formation of the basal network of afferents and efferents necessary for the connection among these three systems, appears uncoupled from the segmentation process.

During neocortical development, network activity undergoes a dramatic transition from largely synchronized, so-called cluster activity, to a relatively sparse pattern around the time of eye-opening in rodents. Biophysical mechanisms underlying this sparsification phenomenon remain poorly understood. Here, we present a dynamic systems modeling study of a developing neural network that provides the first mechanistic insights into sparsification. We find that the rest state of immature networks is strongly affected by the dynamics of a transient, unstable state hidden in their firing activities, allowing these networks to either be silent or generate large cluster activity. We address how, and which, specific developmental changes in neuronal and synaptic parameters drive sparsification. We also reveal how these changes refine the information processing capabilities of an in vivo developing network, mainly by showing a developmental reduction in the instability of network’s firing activity, an effective availability of inhibition-stabilized states, and an emergence of spontaneous attractors and state transition mechanisms. Furthermore, we demonstrate the key role of GABAergic transmission and depressing glutamatergic synapses in governing the spatiotemporal evolution of cluster activity. These results, by providing a strong link between experimental observations and model behavior, suggest how adult sparse coding networks may emerge developmentally.

During neocortical development, network activity undergoes a dramatic transition from largely synchronized, so-called cluster activity, to a relatively sparse pattern around the time of eye-opening in rodents. Biophysical mechanisms underlying this sparsification phenomenon remain poorly understood. Here, we present a dynamic systems modeling study of a developing neural network that provides the first mechanistic insights into sparsification. We find that the rest state of immature networks is strongly affected by the dynamics of a transient, unstable state hidden in their firing activities, allowing these networks to either be silent or generate large cluster activity. We address how, and which, specific developmental changes in neuronal and synaptic parameters drive sparsification. We also reveal how these changes refine the information processing capabilities of an in vivo developing network, mainly by showing a developmental reduction in the instability of network’s firing activity, an effective availability of inhibition-stabilized states, and an emergence of spontaneous attractors and state transition mechanisms. Furthermore, we demonstrate the key role of GABAergic transmission and depressing glutamatergic synapses in governing the spatiotemporal evolution of cluster activity. These results, by providing a strong link between experimental observations and model behavior, suggest how adult sparse coding networks may emerge developmentally.