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The cerebellum is a part of the hindbrain that accounts for more than half of the neurons of the brain. It helps in the integration of sensory information, coordination and motor control. Recent studies indicate that it also has a role in cognitive processes such as attention and processing of language (Ben-Yehudah and Fiez, 2008; Bugalho et al., 2006). The generation of several layers of the cerebellum is a highly intricate and cryptic process. The Purkinje cells, glia and all interneruons are generated in the ventricular zone near the fourth ventricle in a manner similar to what is seen in the developing cerebral cortex. However, in contrast to the pattern mentioned above, the granule cell progenitors arise from a completely different region situated adjacent to the fourth ventricle, called the rhombic lip. These precursors migrate away from the rhombic lip to form a secondary zone of proliferation, on top of the developing Purkinje (PL) and molecular layers (ML). This region is called the External Granular Layer (EGL). The cells in this layer proliferate extensively thereby increasing the layer thickness. Following repeated rounds of division, some cells exit the cell cycle and migrate radially, with the aid of Bergmann glia fibres to form a layer beneath the Purkinje layer. This layer containing mature granule neurons is called the Internal Granular Layer (IGL) (Dahmane and Ruiz i Altaba 1999). Thus the granule neurons precursors generated superficially migrate into the deeper layers of the cerebellum in an 'outside-in' manner.
Although considerable amount of work has been done on cerebellum development in several animal models, very little is known about the mechanisms involved in human cerebellum development. Therefore the first objective of our study was to elucidate the signaling pathways involved in human cerebellum development. Previous studies in mice have implicated sonic hedgehog (Shh) signalling as a major contributor to cerebellum development (Dahmane and Ruiz i Altaba 1999). The obvious step was then to check if the pathway is conserved in humans as well. We carried out the study in cerebellar autopsy samples obtained from Army Base Hospital and the All India Institute of Medical Sciences, New Delhi. Our results indicate the presence of a similar pattern of development as seen during mouse cerebellum development. During the gestational period between the 14th week to term, Shh is detected predominantly in the PL, while its downstream components are detected in the outer EGL. Sonic hedgehog continues to express itself in the PL even postnatally, until the 8-12th month. The decrease in expression coincides with the obliteration of the EGL.
We also looked carefully at the cases of gestational age 10-13 weeks. The histogenic profile seen during this period is unique to humans. During the 10-13th week, there is no conspicuous Purkinje layer, although the EGL is formed. Such a stage is not seen in the rodent model. We found that during the 10-13th week, Shh was produced by the EGL itself, which possibly acts in an autocrine manner. This continued till a distinct PL was formed, following which the Purkinje cells took over the task of Shh release.
This is an important finding in the context of medulloblastomas, the origin of which is not clearly established. Medulloblastomas arise postnatally during a period where the EGL ceases to exist. Why the granule cells continue to proliferate even beyond the 2nd postnatal year, is not clear The origin of medulloblastoma has often been attributed to the granule cell precursors of the External Granular layer and their excessive proliferation (Thomas et al., 2009). Studies have shown that a deregulation in the sonic hedgehog (Shh) pathway induces excessive proliferation of cells of the EGL, ultimately leading to formation of the tumor (Kenney et al., 2003; Thomas et al., 2009). A large number of clues regarding the origin of these tumors have come from genetic studies where mutations of genes involved in the sonic hedgehog (Shh) pathway, like Patched-1 have been implicated (Thomas et al., 2009). However, the reason behind the continual proliferation of EGL cells even in the postnatal cerebellum, after cessation of Shh production by the Purkinje cells, is not known.
We looked at the expression of Shh in classical and desmoplastic medulloblastoma samples where the tumour was accompanied by a portion of the adjoining cerebellum. Our results show that the portion of the cerebellum containing the tumor shows strong expression of Shh and so does the cerebellum adjacent to the tumor that has remnants of EGL which also express Shh, while the Purkinje layer directly below did not stain positive for the same. In contrast, the portion of the cerebellum farthest from the tumor showed no EGL and was comparable with a normal age matched cerebellum. This region too showed no expression of Shh. The PL however continued to express Calbindin. Our results hence seem to indicate that following birth although the EGL does disappear over time, in some individuals, in some regions of the cerebellum EGL proliferation may continue to persist. Persistent autocrine Shh signalling of the EGL or recapitulation of Shh dependent ontogeny, in the postnatal cerebellum could possibly be one of the reasons why medulloblastomas occur. It is hence a befitting example of ontogeny gone wrong, leading to oncogeny.
The human cerebellum undergoes rapid growth in the third trimester (Rakic, 1971; Volpe, 2009). This is in striking contrast to the development of the cerebellum in the commonly seen animal model system, the rodent, in which the cerebellum is relatively immature at birth and the proliferation of the external granule layer (EGL), the formation of the inner granule layer (IGL) and foliation all occur postnatally (Corrales et al., 2006). MRI studies have shown that this rapid growth in the third trimester is impeded by preterm delivery where childbirth occurs at a period less than 37 completed weeks of gestation with resulting lower cerebellar volume (Limperopoulos et al., 2005a; Limperopoulos et al., 2005b; Limperopoulos et al., 2005c; Volpe, 2009). Given the importance of the cerebellum in cognitive functions and the rapid phase of development that occurs in the third trimester there is little information on how the normal developmental program of the cerebellum is modified by change in the environment due to preterm delivery.
The human cerebellum in utero undergoes a clearly defined transition from a 5-layered structure to a mature and anatomically simpler 3-layered structure making the study of developmental alterations at the cellular and molecular level in the cerebellum possible (Rakic and Sidman, 1970; Volpe, 2009). This study addresses the changes at the level of individual cell types that cannot be detected by MRI analysis. To address the issue of how the normal developmental program is perturbed due to premature birth, several morphological parameters and molecular markers were analyzed in the cerebellum of preterm infants who had survived in an ex-utero environment and compared with controls in which the development of the cerebellum has taken place in-utero. The study reports a selective change in the differentiation of granule cells and the Bergmann glia due to the ex-utero environment that could have major consequences for later outcomes.
The last part of my work focuses on the regulation of proliferation and differentiation of granule cell progenitors of the EGL. The major question that we have attempted to address here is how cells of the EGL switch from being proliferative stem cells to differentiated neurons. In the telencephalon a shift from a vertical cleavage (plane of division perpendicular to pial surface) to horizontal plane correlates with an asymmetric (neurogenic) cell division, but whether this is also true in the cerebellum is unknown. There is currently no information as to whether Numb, E-Cadherin and β-catenin are distributed asymmetrically in GCPs as they are during cell divisions in the telencephalon. Our results seem to indicate that while during early cerebellar development (P0-P3) all the markers mentioned above are distributed symmetrically, over time (P5 onwards), the distribution becomes asymmetric. While Beta Catenin is distributed in the apical cell (facing the pial surface), which in all probability will remain a stem cell, Numb and E Cadherin are asymmetrically distributed into the basal cell (facing the Ventricular zone), which will subsequently differentiate into a neuron. This shift in distribution of the markers mentioned also coincides with an increase in percentage of Neuro D positive cells in EGL. We have also studied the distribution of the molecules mentioned above following perturbations in Shh and Wnt signaling pathways. We find that the Shh and Wnt pathways act at the same time to regulate the synthesis and distribution of these molecules. Whether the interaction between these pathways is synergictic or antagonistic is yet to be resolved. The study is important, as it will give us insight on whether pathways governing histogenesis in the cerebral and cerebellar cortex are conserved. It will also help us move a step closer to identifying the molecular switch involved in the conversion of a stem cell into a neuron.