A Novel and Multivalent Role of Pax6 in Cerebellar Development

Abstract

Pax6 is a prominent gene in brain development. The deletion of Pax6 results in devastated development of eye, olfactory bulb, and cortex. However, it has been reported that the Pax6-null Sey cerebellum only has minor defects involving granule cells despite Pax6 being expressed throughout cerebellar development. The present work has uncovered a requirement of Pax6 in the development of all rhombic lip (RL) lineages. A significant downregulation of Tbr1 and Tbr2 expression is found in the Sey cerebellum, these are cell-specific markers of cerebellar nuclear (CN) neurons and unipolar brush cells (UBCs), respectively. The examination of Tbr1 and Lmx1a immunolabeling and Nissl staining confirmed the loss of CN neurons from the Sey cerebellum. CN neuron progenitors are produced in the mutant but there is an enhanced death of these neurons as shown by increased presence of caspase-3-positive cells. These data indicate that Pax6 regulates the survival of CN neuron progenitors. Furthermore, the analysis of experimental mouse chimeras suggests a cell-extrinsic role of Pax6 in CN neuron survival. For UBCs, using Tbr2 immunolabeling, these cells are significantly reduced in the Sey cerebellum. The loss of UBCs in the mutant is due partly to cell death in the RL and also to the reduced production of progenitors from the RL. These results demonstrate a critical role for Pax6 in regulating the generation and survival of UBCs. This and previous work from our laboratory demonstrate a seminal role of Pax6 in the development of all cerebellar glutamatergic neurons.

Significance statement: Pax6 is a key molecule in development. Pax6 is best known as the master control gene in eye development with mutations causing aniridia in humans. Pax6 also plays important developmental roles in the cortex and olfactory bulb. During cerebellar development, Pax6 is robustly expressed in the germinal zone of all glutamatergic neurons [cerebellar nuclear (CN) neurons, granule cells, and unipolar brush cells (UBCs)]. Past work has not found abnormalities in the CN and UBC populations. Our study reveals that the Pax6-null mutation dramatically affects these cells and identifies Pax6 as a key regulator of cell survival in CN neurons and of cell production in UBCs. The present study shows how Pax6 is key to the development of glutamatergic cells in the cerebellum.

Keywords: Atoh1; Pax6; Tbr1 and cerebellar nuclear neurons; Tbr2 and unipolar brush cells; rhombic lips.

Figure 1.

Transcription factors Pax6, Tbr1, and Lmx1a are expressed in the progenitors of CN neurons during cerebellar development. A, The whole-cerebellum transcription profile of Pax6 and Tbr1 during cerebellar development based upon CbGRiTS data. y-axis log₂ transformed and followed by a 2Z+8 Z-score stabilized intensity value for microarray dataset. B, The cerebellum transcription profile of Lmx1a during cerebellar development based upon RIKEN FANTOM5 data. The y-axis shows expression level in tags per million (TPM). C, In the E13.5 cerebellum, immunohistochemistry reveals that Pax6 is expressed in the cells of the subpial stream, which houses newly generated CN neuron progenitors. D, E, Once the progenitors enter the NTZ, Pax6 is rapidly downregulated and now these CN neuron progenitors express Tbr1 (D) and Lmx1a (E). SS, Subpial stream. Error bars represent SE. Scale bars, 100 μm.

Figure 2.

The lack of Pax6 in the Sey-mutant cerebellum results in the absence of RL-derived Tbr1⁺ CN neurons but no apparent loss of VZ-derived Irx3⁺ CN neurons. A, The transcription profile from CbGRiTS of Tbr1 in the wild-type and Sey cerebellum at E13, E15, and E18. The Sey cerebellum has a significant reduction in Tbr1 transcript at E13 and E15. y-axis log₂ transformed and followed by a 2Z+8 Z-score stabilized intensity value for microarray dataset. B–D, Immunohistochemistry reveals that Tbr1⁺ cells are absent in the Sey cerebellum. Cells immunopositive for Tbr1⁺ are observed in the NTZ of the wild-type cerebellum (white arrows) at E13.5 (B), E15.5 (C), and E18.5 (D), but this Tbr1⁺ cell population is absent in the Sey cerebellum (red arrows). A few Tbr1⁺ cells are observed in the lateral cerebellum of the E15.5 Sey mutant (C, blue arrowhead). These cells comprise only 2.7% (±0.4%) of the total number of Tbr1⁺ cells in the wild-type cerebellum. E, Immunohistochemistry of Tbr1 illustrates that the cerebellar expression of Tbr1 in heterozygous (Pax6^Sey/+) at E13.5, E15.5, and E18.5 is not different from that of the wild-type littermates (B–E, compare white arrows). F, Immunohistochemistry of Irx3 over developmental time reveals that these CN neuron populations are similar in the cerebellum of the wild-type (top) and Sey (bottom) mouse at E11.5, E13.5, and E15.5. At E11.5, the Irx3⁺ CN neurons are largely seen outside of the VZ. By E13.5, Irx3⁺ CN neurons have entered the NTZ. By E15.5, the Irx3⁺ CN neurons have started to colonize the cerebellar core. Error bars represent SE. Scale bars, 100 μm.

Figure 3.

The lack of Pax6 in the Sey cerebellum results in the reduction of Lmx1a⁺ cells in the cerebellar nuclei. A, B, Lmx1a expression is observed in CN neuron progenitors that enter the NTZ of (A) E13.5 and (B) E15.5 wild-type cerebellum (white arrows). In the E13.5 and E15.5 Sey cerebellum, Lmx1a⁺ cells are largely absent (red arrows). The lateral aspects of the cerebellum highlight the c3 cells (B, bottom, white arrowheads) which are generated from the VZ and give rise to extracerebellar neurons. These cells also express Lmx1a and are not altered in the Sey mutant. Scale bars, 100 μm.

Figure 4.

Cytoarchitecture of the Sey cerebellum indicates the loss of CN neurons and UBCs. A, Cresyl violet staining reveals the aggregation of CN neurons at the NTZ in the E15.5 wild-type cerebellum (black arrow). B, In contrast, this nuclear mass is replaced by a fibrous and acellular matrix in the Sey cerebellum (red arrow). C, D, In the E18.5 cerebellum (C), the CN neurons have descended from the NTZ in the wild-type cerebellum (black arrow), while this nuclear mass is absent in the Sey cerebellum (D, red arrow). At the medial level, UBCs are found in the RL region of wild-type cerebellum (C, bounded by red dotted line), but the same region is devoid of cells in the Sey cerebellum (D, bounded by red dotted line). E, F, UBC progenitors (bounded by red dotted lines) are found in wild-type (E) and Sey (F) lateral cerebella. Scale bars, 100 μm.

Figure 5.

CN neuron progenitors are generated in the Sey-mutant cerebellum but exhibit enhanced cell death. A, In the E11.5 wild-type (Pax6^+/+; Atoh1^+/LacZ) cerebellum, CN neuron progenitors express Atoh1 when the cells emerge from the RL and during migration along the subpial stream (arrows). B, A similar pattern of Atoh1-expressing cells is observed in the Sey-mutant (Pax6^sey/sey; Atoh1^+/LacZ) cerebellum (arrows). C–E, Cell death is assessed by immunolabeling for activated caspase-3 and quantified at E12.5–E18.5 in wild-type and Sey cerebella. C, D, The majority of cells undergoing cell death in the wild-type and Sey cerebellum are observed at the anterior of the EGL (arrowheads). D, There is a dramatic increase in caspase 3⁺ cells in the mutant EGL. E, The Sey cerebellum shows significantly higher numbers of anti-caspase-3⁺ cells than that in the wild-type at E13.5 and later (*p < 0.005). F–H, Double-labeling with Insm1, a GC-specific marker, and caspase-3 reveals that most of the dying cells in the Sey cerebellum are non-GCs. GCs robustly expressed Insm1 in the wild-type (F) and Sey (G) EGL. Highlighted area in G is shown in H at higher magnification. H, In the Sey EGL, most caspase-3⁺ cells are Insm1-negative (blue arrows) and few cells coexpress Insm1 and caspase-3 (yellow arrows). I, Proportion of caspase-3⁺ cells that are Insm1-positive (pink) or Insm1-negative (blue) in the E13.5 and E15.5 wild-type and Sey EGL. The Sey cerebellum exhibits a significantly increased number of total anti-caspase-3⁺ cells than the wild type at both E13.5 and E15.5 (*p < 0.005). SS, Subpial stream. Error bars represent SE. Scale bars, 100 μm.

Figure 6.

Experimental chimera analysis demonstrates that Pax6 can be cell-extrinsic for CN neuron survival. Experimental chimeras were examined at E18.5. Three embryos (H, I, and O) with cellular contributions from the Sey/Sey genotype are examined for the phenotype of CN neuron survival. A, Quantitative analysis of the number of Tbr1⁺ cells in wild-type, Sey-mutant, and Sey-chimeric cerebella. Expected numbers of Tbr1⁺ cells for each chimera are calculated from the percentage chimerism and the Tbr1⁺ cell counts from wild-type and Sey cerebella. A χ² test reveals that the observed numbers of Tbr1⁺ cells in the chimeric cerebella are significantly different from the expected numbers. B, Cells positive for Tbr1 expression are localized to the medial part of the nuclear region (shown in box). Higher magnification of the area in the box is shown in C and D. C, Costaining with phenotype marker (Tbr1) and genotype marker (GFP) reveals that Tbr1⁺ cells in the chimeric cerebellum arise from both wild-type (GFP⁺ and Tbr1⁺; arrows) and Sey/Sey-mutant (GFP⁻ and Tbr1⁺; arrowheads) genotypes. D, This image more clearly shows the mutant Tbr1⁺ cells (arrowheads) and wild-type Tbr1⁺ cells (arrows). CP, Choroid plexus. Error bars represent SE. Scale bars, 100 μm.

Figure 7.

The loss of Pax6 results in the absence of Tbr2⁺ cells from the medial Sey cerebellum. A–C, In the normal E15.5 cerebellum, immunohistochemistry reveals that Pax6 (A), Lmx1a (B), and Tbr2 (C) label a stream of cells (within the dashed lines) found between the EGL and the interior face of the RL. D, The transcription level of Tbr2 in the Sey cerebellum is significantly reduced at E15.5 and E18.5 as revealed by Pax6 transcriptome analysis from CbGRiTS. y-axis log₂ transformed and followed by a 2Z+8 Z-score stabilized intensity value for microarray dataset. E, F, In the E18.5 wild-type cerebellum, expression of Tbr2 is observed in the UBCs moving out of the RL into the developing cerebellar core (arrows). G, In contrast, Tbr2⁺ cells are absent from the RL and cerebellar core of the Sey cerebellum at medial levels. H, Some Tbr2-positive cells are observed more laterally in the Sey cerebellum (arrows). CP, Choroid plexus. Error bars represent SE. Scale bars, 100 μm.

Figure 8.

The loss of Pax6 results in a reduction of Lmx1a⁺ cells in the Sey cerebellum. A, At E15.5, UBC progenitors with strong Lmx1a expression (left, white arrowheads) are missing in the Sey cerebellum (right). By contrast, cells with light Lmx1a expression, which are GC progenitors born about the same time as UBCs, have similar staining in cells of the wild-type and Sey RL. B, At E18.5, cells with Lmx1a expression in the wild-type cerebellum (left, white arrowheads) are absent from the Sey medial cerebellar core (right). Lmx1a⁺ cells are seen in the wild-type, lateral cerebellum (bottom left, white arrowheads), which are also observed in lateral portions of the E18.5 Sey cerebellum (bottom right, white arrowheads). CP, Choroid plexus. Scale bars, 100 μm.

Figure 9.

Enhanced cell death and decreased neurogenesis leads to the reduction of UBCs in the Sey cerebellum. A, Activated caspase-3 immunopositivity indicates the absence of apoptotic cells in the E16.5 wild-type cerebellum while caspase-3⁺ cells are found in the RL of E16.5 Sey cerebellum (arrowhead). B, Quantitative analysis of caspase-3 immunopositivity revealed a significant increase in cell death in the Sey cerebellar RL at E16.5 compared to the wild type (*p < 0.005). C–E, The RL (C, area bounded by dotted lines) of E16.5 wild-type and Sey cerebella demonstrates the mutant RL is significantly smaller (D) and has significantly fewer BrdU⁺ cells (E) compared with the wild type (*p < 0.005). F, Interestingly, the percentage of BrdU⁺ cells in the Sey RL is slightly higher than that in the wild type. Error bars represent SE. Scale bars, 100 μm.

Figure 10.

A model molecular program underpinning the development of cerebellar glutamatergic neurons. Expression of Atoh1 in the RL is required to specify progenitor cells to RL lineages. Pax6 is subsequently expressed in all RL-derived glutamatergic neurons and regulates multiple developmental processes in these cell types. In the development of CN neurons, Pax6 regulates cell survival of progenitors. The present study identifies an enhanced cell death in the Pax6-null cerebellum that contributes to the loss of CN neurons. It is known that expression of Tbr1 regulates the migration of CN neurons and we find this is downstream of Pax6 function. In the development of UBCs, the survival and production of UBC progenitors require Pax6 function. Thus, in the Pax6-null mutant there is an enhanced cell death and reduction in UBC progenitor cells, which results in a reduction of UBCs. Tbr2 functions downstream of Pax6, and plays a role in UBCs migration. In the development of granule cells, the current findings suggest that Pax6 plays a role in cell survival and cell proliferation, in addition to cell differentiation and migration. Our current work also suggests an earlier function of Pax6 in regulating the replenishment of the RL progenitor pool.