Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression

Abstract

We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1a(Cre/Cre) mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

Fig. 1.

Ptf1a locus activity and expression in the developing cerebellum. (A) Lineage tracing analysis of Ptf1a activation in the cerebellum of Ptf1a^Cre/+;R26R embryos revealed by X-Gal staining. In toto labeling showing strong X-Gal staining in the cerebellar plates (arrows) of E12 embryos. (B and C) Ptf1a mRNA expression in the cerebellum. ISH for Ptf1a mRNA. (B) At E12, the hybridization signal was detected (arrows) at the proliferating VZ and in individual migrating neurons. (C) At E18, the hybridization signal was detected in scattered single cells (arrows) throughout the cerebellar parenchyma. (D–G) Immunohistochemical expression of Ptf1a. (D) Ptf1a localized within the VZ and in migrating postmitotic cells (arrows) at E14. (E) At E16, a similar pattern of staining of migrating cells is observed. (F) At P5, migrating interneurons expressing Ptf1a (arrows) were observed below the PCL, whereas PCs and granular cells were nonreactive. A Ptf1a-positive interneuron having reached the molecular layer (ML) is identified with an arrowhead. (G) Ptf1a was not detected in the cerebellum of P15 mice. [Scale bars: (A) 50 μm; (B) 50 μm, represents 70 μm in C; (D) 50 μm, pertains to E and represents 100 μm in F and G.]

Fig. 2.

Cerebellar histology in wild-type and Ptf1a null E18 embryos. (A and B) Coronal sections immunostained for Calb. At E18, the Calb-positive PCL (arrows in A) was absent in Ptf1a null embryos (B). (C–F) Immunolabeling for the PC markers ROR-α and Dab1 at E18. Note that ROR-α and Dab1-positive PCs were absent in sections from Ptf1a null embryos. (G and H) ISH for GAD65/GAD67 mRNA showed a dramatic reduction of expression in the mutant cerebella at E18. (I and J) Sagittal sections immunostained for Math1 in E18 wild-type (I) and Ptf1a null (J) embryos. As in wild-type embryos, cells in the EGL of Ptf1a-deficient mice express the typical granule cell marker Math1. [Scale bars: (A) 100 μm, pertains to B; (C) 50 μm, pertains to D–F; (G) 100 μm, pertains to H–J.]

Fig. 3.

Lineage tracing of Ptf1a-expressing cells and contribution of E12-generated cells to the EGL. (A and B) Lineage tracing of Ptf1a-expressing cells and contribution to the EGL. In Ptf1a^Cre/+;R26R mice, cells with an activated Ptf1a locus do not contribute to the EGL (A). By contrast, the EGL, but not the RL, of Ptf1a null embryos is densely populated by cells with an activated Ptf1a locus (B). (C and D) In wild-type mice, BrdU-positive cells are found in the region of the PCL and in the parenchyma but not in the EGL (C). In contrast, Ptf1a-deficient mice display a very high number of BrdU-positive cells in the EGL (D, arrows). (E–H) In Ptf1a^Cre/+;R26R mice, labeled cells are found mainly in the PCL and are absent from the EGL (E). In Ptf1a^Cre/Cre;R26R cerebella, labeled cells are abundant in the EGL and in the deep layers (F). Double immunostaining with antibodies detecting β-Gal showed the presence of BrdU-labeled cells that had activated the Ptf1a locus in the EGL (F–H). [Scale bars: (A) 100 μm, pertains to B; (C) 100 μm, pertains to D; (E) 100 μm, pertains to F–H.] The EGL is labeled by dashed lines.

Fig. 4.

Cerebellar ventricular zone progenitors lacking Ptf1a acquire an EGL-like cell phenotype at E18. (A–F) Double-labeling immunofluorescence with antibodies detecting β-Gal and the granule cell marker Reelin in the cerebellum of Ptf1a^Cre/+;R26R and Ptf1a^Cre/Cre;R26R embryos. In wild-type cerebella, cells with an activated Ptf1a locus do not express Reelin and display characteristic distribution of PCs and interneurons (A–C). In mutant cerebella, β-Gal-positive cells are found in the rostral EGL (large arrowheads) and coexpress Reelin (D–F, arrows). (G–L) Double-labeling immunofluorescence with antibodies detecting β-Gal and Zic1/2 in the cerebellum of Ptf1a^Cre/+;R26R and Ptf1a^Cre/Cre;R26R embryos. In wild-type cerebella, Ptf1a-derived cells do not express Zic1/2 and display the distribution of PCs and interneurons (G–I). In mutant cerebella, β-Gal-positive cells in the rostral EGL (large arrowheads) coexpress both β-Gal and Zic1/2 (J–L, arrows). (M–O) β-Gal-positive cells located in the EGL (large arrowheads) of Ptf1a^Cre/Cre;R26R mutant embryos are also labeled by Math1 antibodies (arrow). [Scale bars: (A) 50 μm, pertains to B–F and J–L; (G) 50 μm, pertains to H and I; (M) 25 μm, pertains to N and O.]

Fig. 5.

Proliferation and ultrastructure of Ptf1a-derived cells in Ptf1a^Cre/Cre;R26R embryos. (A–F) Pregnant females were injected with BrdU at E18, and proliferating precursors were analyzed 2 h after the injection. VZ progenitors lacking Ptf1a (β-Gal) and populating the EGL are colabeled by BrdU. Double-labeled β-Gal/BrdU-positive cells in the EGL layer are marked by arrows. (G–J) Electron microscopy of β-Gal-expressing cells in Ptf1a^Cre/Cre;R26R null mutant and Ptf1a^Cre/+;R26R embryos. (G and H) Semithin sections from a Ptf1a^Cre/Cre;R26R embryo illustrating that endogenous (labeled by asterisk) and Ptf1a-derived (Bluo-Gal-stained, labeled by arrows) EGL cells have similar sizes. (I) Electron micrograph showing that Bluo-Gal-labeled cells (arrows) display a fine structure identical to that of unlabeled EGL cells (asterisk). Electron micrograph illustrating a β-Gal-labeled PC in Ptf1a^Cre/+;R26R cerebella. Arrows in J label enzymatic reaction end product. [Scale bars: (A) 150 μm, pertains to B and C; (D) 50 μm, pertains to E and F; (G) 25 μm; (H) 10 μm; (J) 2 μm, pertains to I.]

Fig. 6.

Role of Ptf1a in neuronal specification in the cerebellum. (A) In wild-type mice, Ptf1a-expressing VZ progenitors produce GABAergic cells in the cerebellum, including PCs and interneurons (red). In parallel, RL progenitors produce granular cell precursors located in the EGL expressing Math1, Reelin, and Zic1/2 (blue). (B) In absence of Ptf1a, VZ precursors are unable to produce functional GABAergic cells but generate small-sized cells expressing Math1, Reelin, and Zic1/2, which abnormally invade the EGL (green). In Ptf1a null embryos, the production of granule cells by RL progenitors is preserved. Thereby, the EGL of Ptf1a null embryos is populated in part by EGL cells produced normally in the RL (blue) and by cells with an EGL-like phenotype produced by VZ progenitors lacking Ptf1a (green).