In this study, histological results reveal that the overall size and folium structure of the cerebellum, as well as the relative portions of different cerebellar cortical layers, are altered in TR4−/− mice during postnatal development. In addition to histological differences between TR4−/− and TR4+/+ cerebella, impaired motor coordination is apparent in TR4−/− mice at the age of 2 weeks and persists into adulthood. These changes in cerebellar cytoarchitecture and this abnormal behavior in postnatal TR4−/− mice suggest that TR4 may play an important role in cerebellar development.
Further analysis of the developing TR4−/− cerebellum supports the idea that alterations in cerebellar cytoarchitecture, especially the reduction in IGL density and size, may result from a combination of effects including disturbances in the proliferation of granule cell progenitors, delayed inward migration of postmitotic granule cells, and higher apoptotic incidence. Intriguingly, the proliferation of granule cell progenitors in the TR4−/− EGL exhibits a pattern opposite that in the EGL of the control TR4+/+ cerebellum during postnatal development. In the TR4+/+ EGL, the proliferation of granule cell progenitors increases during the first postnatal week and then declines to undetectable levels during the second and third postnatal weeks. However, in the TR4−/− EGL, fewer proliferation signals can be detected at P0 and P7 than in the control EGL. Conversely, a prolonged period of granule cell proliferation occurs. In TR4−/− mice, proliferating granule cells can be found at P14 and even as late as P18, while no proliferation can be detected in the controls at these ages. This shift in the proliferation pattern suggests that TR4 may have different functions depending on developmental time. Before P7, the expression of TR4 may be essential for the proliferation of granule cells. However, after P7, TR4 may be involved in controlling exit from the cell cycle. Such a switch in the function of TR4 in granule cell proliferation could arise from changes in the composition of participating coregulators or other nuclear receptors. Previous in vitro studies have demonstrated that TR4 has the ability to cross talk or form dimer transcriptional complexes with numerous other nuclear receptors. In addition to the abnormal proliferation pattern of granule cells in the TR4−/− EGL, delayed migration of postmitotic granule cells and higher incidences of apoptosis in both the ML and IGL are observed in the TR4−/− cerebellum. Thus, granule cell proliferation is prolonged in the TR4−/− EGL, but the subsequently differentiated granule cells fail to migrate into the IGL at the appropriate time. This failure to reach the correct location at the proper time may induce programmed cell death in these postmitotic granule cells, resulting in reduced width and cellular density of the TR4−/− IGL. This interpretation is supported by the significant reduction in the number of BrdU-labeled cells in the TR4−/− IGL and ML 72 h after administration, during which time cells are normally evenly distributed in the control IGL. Taken together, prolonged proliferation and slower migration of granule cells in the TR4-deficient cerebellum can explain the delayed disappearance of the EGL during the late development stages (P14 to P21) of TR4−/− mice.
In addition to the abnormalities observed in granule cells, dendritic arborization of Purkinje cells in the TR4−/−
cerebellum is considerably stunted, and the labeling intensity of calbindin is significantly reduced, during the first two postnatal weeks. These findings further suggest that Purkinje cell differentiation or function may be compromised in the cerebella of TR4-deficient mice early in postnatal development. It has been demonstrated that when postmitotic granule cells depart from the inner portion of the EGL and migrate radically into the IGL, their axons extend horizontally to establish synapses with the dendrites of Purkinje cells. During this process, the ML grows considerably in size to accommodate the extensions of neurites of both cell types. Thus, the stunted formation of dendritic trees of Purkinje cells and the delayed migration of granule cells indirectly suggest that the extension of neurites of both Purkinje and granule cells is affected and may account for the reduced width of the ML in the TR4−/−
cerebellar cortex. During cerebellar development, the interaction between Purkinje and granule cells guides other aspects of their cell survival and behavior (11
). Although expression of TR4 can be detected in Purkinje and granule cells in the normal postnatal cerebellum, in the present model we are not able to determine whether the alterations in granule cell proliferation and migration, as well as Purkinje cell development, are cell autonomous. These effects may reflect abnormal interactions between Purkinje and granule cells, due to deficits in function of either or both cell types. However, examination of the number and distribution of basket and stellate cells in the ML, as well as of Golgi cells in the IGL, reveals no obvious differences in these inhibitory interneurons between TR4−/−
and age-matched TR4+/+
mice. Thus, it is likely that TR4 is specifically involved in the development of granule and Purkinje cells, which in turn affects the cerebellar structures they populate.
Interestingly, morphological alterations occur in the CNS even earlier, at embryonic stages of the TR4−/−
cerebellum. This finding is consistent with a previous study showing the predominant expression of TR4 in the mesencephalon at the embryonic stage, which is believed to be the origin of cerebellar progenitors (37
). In the TR4−/−
embryo, the size of the rhombic lip and the thickness of cell layers in the neuroepithelium are reduced, while no obvious difference appears in the structure of other organs relative to those of control littermates. As revealed by BrdU incorporation assays in TR4−/−
embryos, the reduced number of cells in these two germinal zones results from interference with the proliferation of these progenitors. At E18.5, the expression of Zic1 and Pax-6, which have been suggested to play fundamental roles in the neurogenesis of cerebellar granule cells (2
), increased 1.6- and 1.8-fold, respectively, in the TR4−/−
cerebellum over that in controls. This result suggests that during the commencement of cerebellar development, some compensatory processes may be triggered to overcome the effects of losing TR4. Thus, our results from the TR4−/−
embryo further demonstrate that TR4 is important very early in neurogenesis.
Previous studies with staggerer (sg) and RORα−/−
mice, which express deficits in the thyroid hormone/RORα signaling pathway, revealed a cell-autonomous defect of the Purkinje cells. Purkinje cells of sg and RORα−/−
mice fail to establish synaptic contacts with granule cell parallel fibers, causing granule cell numbers to decrease. In addition, the proliferation and migration of granule cells are disrupted in sg and RORα−/−
mice. Both types of mutant mice exhibit perturbed cerebellar development and present with tremor and impaired balance (8
). The abnormalities in sg and RORα−/−
mice are similar to those we observed in TR4−/−
mice. Because TR4 is able to modulate thyroid hormone and retinoic acid signaling (21
), it is possible that the defects observed in TR4−/−
cerebellar structure may result, in part, from disturbed thyroid hormone signaling. However, the aberrant development of Purkinje cells in the TR4−/−
cerebellum is distinct from that in sg and RORα−/−
mice. Although the growth of dendritic branches of Purkinje cells is stunted in the cerebella of all three types of mice, only sg and RORα−/−
mice show a reduction in the number of Purkinje cells at late developmental stages. In contrast, the Purkinje cell dendrites in the TR4−/−
cerebellum can extend into the ML at late developmental stages, although the thickness of the primary bundle and the length of dendritic trees are obviously reduced relative to those of the TR4+/+
cerebellum. To determine whether thyroid hormone signaling genes are affected, we examined the expression of the RORα gene, which has been implicated as the major effector for thyroid hormone signaling in cerebellar development. RORα gene expression in the TR4−/−
cerebellum is reduced to 72.92% of the level in TR4+/+
controls at P7, when the TR4−/−
cerebellum shows a pattern of disorganization most similar to that of thyroid hormone-deficient mice. Interestingly, at E18.5, RORα expression is up-regulated 1.3-fold in the TR4−/−
cerebellum. Thus, we surmise that during cerebellar development, TR4 may cross talk with RORα in regulating the expression of some genes that are essential for cerebellar development. The fact that TR4−/−
mice survive beyond the age of 4 weeks, when most sg and RORα−/−
mice die, suggests that other genes may compensate for the deficiency of TR4.
Besides the thyroid hormone deficiency model, several other null mice models have been studied in order to understand neurogenesis. TR4-deficient mice also share several similarities in abnormal cerebellar development with those mutant mice, including defects in the proliferation and migration of granule cells and in the development of Purkinje cells. Therefore, we further examined those well-documented genes which have been suggested to be involved in cerebellar development in order to gain insight into the physiological function of TR4 in neurogenesis. Previous studies with reeler and Cdk-5−/−
mice have shown that disturbances in the arrangement of Purkinje cells cause a failure in cerebellar development (26
). Related studies further demonstrated that reelin, the mutant gene in reeler mice, is critical for neuronal migration and cell position in the CNS (25
). In the reeler cerebellum, Purkinje cells fail to form an appropriate plate structure to support the proliferation of granule cells in the EGL, resulting in reductions in the size and foliation of the cerebellum (27
). The disturbed arrangement of Purkinje cells in the neonatal TR4−/−
cerebellum, and the ability of TR4 to regulate the expression of ApoE, whose receptor has been shown to be involved in the degradation of neuronal adaptor protein Disabled-1, the key molecule in reelin signaling (4
), led us to hypothesize that reelin signaling may be affected. Consistent with this hypothesis, reelin expression is reduced in the TR4−/−
cerebellum at different postnatal stages. Interestingly, in reeler mice, although the Purkinje cells are disoriented, no disturbance is found in granule cell migration. This result is distinct from our observation for the TR4−/−
cerebellum. Moreover, at later developmental stages, the Purkinje cells in the TR4−/−
cerebellar cortex seem to align normally, although severely stunted dendritic arborization occurs at P7. Cdk-5 has been demonstrated to be important in neurogenesis, specifically for neuronal migration and neurite outgrowth (17
). Based on the stunted Purkinje cell arborization and the deficit in granule cell migration in the postnatal TR4−/−
cerebellum, we predicted that the expression of Cdk-5 may also be altered. As revealed by real-time RT-PCR and immunohistochemistry, the expression level of Cdk-5 is reduced, specifically in Purkinje and granule cells. Thus, the present data suggest that the abnormal development of Purkinje cells in the TR4−/−
cerebellum may result in part from impaired reelin and Cdk-5 signaling. In the cerebellum, in addition to making direct synaptic contacts with granule cells, Purkinje cells also secrete mitogenic factors, such as Shh, to stimulate the proliferation of granule cell progenitors in the secondary proliferative zone, the EGL, and may be involved in guiding the migration of postmitotic granule cells inward (31
). In the postnatal TR4−/−
cerebellum, the development of Purkinje cells is stunted and the level of Shh is decreased at postnatal stages (P0 and P7) relative to those in the TR4+/+
cerebellum. These results further support the idea that the function of Purkinje cells is compromised when TR4 is absent, and they may explain the diminished proliferation of granule cell progenitors in the TR4−/−
EGL at early developmental stages.
It is known that proper inward migration of postmitotic granule cells plays an important role in cerebellar organogenesis. In the developing TR4−/−
cerebellum, granule cell proliferation and Purkinje cell differentiation are affected. Moreover, the profile of granule cell migration from the EGL to the IGL is interrupted. A previous study with Cdk-5-null mice indicated that Cdk-5 is not only essential for Purkinje cell orientation but also important for granule cell migration (30
). The reduced Cdk-5 expression in TR4−/−
granule and Purkinje cells implies that the delayed migration of neuronal cells may be cell autonomous and that TR4 may play a role in regulating Cdk-5 expression in neurons. On the other hand, expression of Pax-6, which has been demonstrated to be important for postmitotic granule cell migration and the formation of parallel fibers (9
), is first increased and then decreased in the TR4−/−
cerebellum. The observation of a lower number of progenitors at the primary proliferative zone but with higher expression of Pax-6 and reelin at embryonic and neonatal stages in the TR4−/−
cerebellum suggests that these mechanisms may be involved in compensating for the lack of TR4 in neurogenesis rather than retarding cerebellar development. Abnormal granule cell migration may also reflect disrupted establishment of neuronal-glial contact during neurogenesis. Astn, a neuronal-glial ligand required for normal migration of neuronal cells (1
), is down-regulated in the TR4−/−
cerebellum at early developmental stages. This raises the possibility that the microenvironment between the granule cells and glial fibers may also be altered in the TR4−/−
cerebellum. Thus, TR4 may play a role in neuronal migration in two ways: regulating the expression of reelin, Cdk-5, and Pax-6 in cerebellar neurons and establishing the proper contact in neuronal-glial circuitry.
Interestingly, the defects in the TR4−/−
cerebellum are similar, but not identical, to those found in several mouse models which concern cerebellar development, including stagger, RORα−/−
, reeler, Cdk-5−/−
, small eye, and astn−/−
). Furthermore, the expression of some of these genes is not simply reduced in TR4−/−
mice. An initial increase in expression is observed for Pax-6 and reelin at embryonic stages (Table ), and expression subsequently declines to below normal levels during later postnatal development. These results implicate TR4 in differentially modulating the expression of genes required for the development of cerebellar neurons, either directly or indirectly. Abolishing TR4 function alters granule cell proliferation and migration, influences Purkinje cell development, and/or affects cytoskeletal organization between glia and migrating granule cells, which is required for proper cerebellar development. Although the expression of genes which are important for cerebellar development is altered in the TR4-deficient cerebellum, the specific cellular and molecular mechanisms underlying these changes still need to be determined. Immunocytochemically, the TR4 signal is observed ubiquitously in normally developing cerebella (Fig. ). Specifically, TR4 is most highly expressed in postmitotic granule cells in the EGL, ML, and IGL, as well as in Purkinje cells. The presence of TR4 in these cells raises the possibility that the abnormalities in EGL and IGL thickness, increased numbers of rounded granule cells in the ML, and stunted growth of Purkinje cell dendritic trees in the TR4−/−
cerebellum may be specifically due to the absence of functional TR4 in these cells. Therefore, it would be interesting to study conditional or cell-specific TR4-null models in order to further clarify the function of TR4 in neurogenesis.
In conclusion, our results show that multiple abnormalities observed in the developing TR4−/− cerebellum may be due to the loss of TR4 function in the CNS. However, whether the cerebellar defects are direct effects of deletion of the functional TR4 gene specifically in Purkinje and/or granule cells or indirect effects due to loss of TR4 in other cell types remains to be determined. Most likely, multiple mechanisms exist by which TR4 regulates the development of the cerebellum, as suggested by our findings. Our results provide important insights into the physiological role of TR4 during cerebellar development and may serve as the basis for further exploration of the function of TR4 in CNS development.