Figure 1. Conditional deletion of Atoh1 from the En1 domain prevents proper size expansion, lamination, and foliation of the cerebellum.

(A) Schematic of an embryonic brain. Inset is the cerebellar anlage. Atoh1 domain (excitatory lineage neurons, including granule cell precursors, pink), Ptf1a domain (inhibitory lineage neurons, including Purkinje cell precursors, green), En1 domain (gray). Orientation is the same for all panels unless otherwise indicated. (B) Schematic of a sagittal section of a P14 cerebellum (left). Cerebellar lobules are labeled with Roman numerals (Larsell, 1952). Schematic of the cerebellar microcircuit with Purkinje cells and granule cells (right). Purkinje cell = green; granule cell = pink; ml = molecular layer, pcl = Purkinje cell layer; gcl = granule cell layer. (C) Intersectional labeling of En1;Atoh1 domain with tdTomato (pink) shows no overlap with Purkinje cells (Calbindin; green). (D) Whole brain images of cerebellum from anterior (left two images) and dorsal view (right two images) from P7 and P14 in control animals. The cerebellum is pseudo-colored in red. (E) Whole brain images of the cerebellum from anterior (left two images) and dorsal views (right two images) from P7 and P14 En1^Cre/+;Atoh1^fl/- mice. The cerebellum is pseudo-colored in red. (F) Outline of cerebellar size in control and En1^Cre/+;Atoh1^fl/- mice at P7 and P14 showing size expansion in control, but not En1^Cre/+;Atoh1^fl/- mice. (G) Sagittal sections of the hindbrain from control mice stained with cresyl violet to visualize gross architecture and regional organization of the brain. (H) Sagittal sections of the hindbrain from En1^Cre/+;Atoh1^fl/- mice stained with cresyl violet showing the general deformities in regional patterning. (I) Outline of cerebellar size in control and En1^Cre/+;Atoh1^fl/- mice at P7 and P14 showing size expansion in control, but not En1^Cre/+;Atoh1^fl/- mice. (J) Sagittal (top) and coronal (bottom) whole section images of cerebella from P7 (left) and P14 (right) control mice. Purkinje cells are stained with Calbindin (green) and DAPI (pink). Note the layered and foliated structure of the cerebellum. (K) Sagittal (top) and coronal (bottom) whole section images of cerebella from P7 and P14 En1^Cre/+;Atoh1^fl/- mice. Purkinje cells are stained with Calbindin (green) and DAPI (pink). All images are presented at the same magnification. Note the lack of layers and foliation. All images in J and K are presented at the same magnification. (L) Outline of cerebellar size in control and En1^Cre/+;Atoh1^fl/- mice at P7 and P14 showing size expansion in control, but not in the En1^Cre/+;Atoh1^fl/- mice. All images are representative of N=3 per age and genotype group.

Figure 1—figure supplement 1. Conditional deletion of Atoh1 from the En1 domain leads to cerebellum-specific morphological abnormalities.

(A) Whole brain sagittal tissue sections from control mice stained using cresyl violet demonstrate the typical anatomical features found throughout the forebrain, midbrain and hindbrain. (B) Whole brain sagittal tissue sections of En1^Cre/+;Atoh1^fl/- mice stained using cresyl violet display cerebellar-specific gross morphological abnormalities. Regions labeled in the control and En1^Cre/+;Atoh1^fl/- brain sections: olfactory bulb, hippocampus, striatum, thalamus, pons, vestibular nuclei, inferior olive, and neocortex. All tissue sections were taken from P14 mice.

Figure 1—figure supplement 2. Conditional deletion of Atoh1 from the En1 domain reduces the density of excitatory cerebellar cell types, but increases the density of inhibitory cerebellar cell types.

(A) En1^Cre/+;Atoh1^fl/- mice lack differentiated granule cells, identified with GABARα6. (B and C) En1^Cre/+;Atoh1^fl/- mice have a reduction in unipolar brush cells, identified by Calretinin and Tbr2. (D) En1^Cre/+;Atoh1^fl/- mice have dense staining for NFH that marks Purkinje cells and excitatory nuclei (interposed nucleus shown here). (E through H) En1^Cre/+;Atoh1^fl/- mice have a high density of inhibitory neurons, revealed with the expression of RORα (E), HCN1 (F), Neurogranin (G), and PV (H). All images are representative for N=3 brains for each genotype and were performed in P14 mice.

Figure 1—figure supplement 3. Conditional deletion of Atoh1 from the En1 domain reduces the density of En1;Atoh1 lineage neurons.

(A) Genetic strategy to express TdTomato in the En1;Atoh1 lineage of control mice. (B) Genetic strategy to express TdTomato in the En1;Atoh1 lineage of conditional knockout mice. (C) Expression of TdTomato in sagittal tissue sections taken at the midline (vermis) and more medial (para-vermis) regions of cerebella from control (bottom, left) and conditional knockout (top, right) mice. (D) High magnification of TdTomato signal in lobule IX of control mice (i and iii) and the ventro-caudal region of conditional knockout mice (ii and iv) showing signal saturation in control mice and individually countable cells (arrows) in the conditional knockout mice.

Figure 1—figure supplement 4. Conditional deletion of Atoh1 from the En1 domain reduces the number of unipolar brush cells and excitatory nuclei neurons.

(A) Co-expression of Tbr2 (green) and TdTomato (pink) in the En1;Atoh1 lineage (as in Figure 1—figure supplement 3) in control (top) and conditional knockout (bottom) cerebellums. (B) Co-expression of NFH (green) and TdTomato (pink) in the En1;Atoh1 lineage (as in Figure 1—figure supplement 3) in control (top) and conditional knockout (bottom) cerebellums. (C) Whole section image of Tbr2 (grey) expressing cells in cerebellum from control mouse. Cerebellar lobules are labeled with Roman numerals (Larsell, 1952). Insets (i, ii, iii) show lower density of Tbr2⁺ cells in dorsal lobule VI (i) than in ventral lobules IX (iii) and X (ii). (D) Whole section image of Tbr2 (gray) expressing cells in the cerebellum from an En1^Cre/+;Atoh1^fl/- mouse. Insets (i, ii) show higher density of Tbr2⁺ neurons in ventral cerebellum (i) than in dorsal cerebellum (ii). (E) Quantification of Tbr2⁺ cells (unipolar brush cells) in control and En1^Cre/+;Atoh1^fl/- mice. (F) NFH (pink) and Calbindin (green) staining in the cerebellum of a control mouse. Insets show examples of NFH⁺/Calbindin^- (i, excitatory nuclei cells) and NFH⁺/Calbindin⁺ (ii, Purkinje cell). In insets, N = NFH signal; C = Calbindin signal; merge = both. (G) NFH (pink) and Calbindin (green) staining in the cerebellum of an En1^Cre/+;Atoh1^fl/- mouse. Insets show examples of NFH⁺/Calbindin^- (i, putative excitatory nuclei cell) and NFH⁺/Calbindin⁺ (ii, Purkinje cell). In insets, N = NFH signal; C = Calbindin signal; merge = both. (H) Quantification of NFH⁺/Calbindin^- cells (putative excitatory nuclei cells for the mutant) in control and En1^Cre/+;Atoh1^fl/- mice. For (E and H) bar height represents the mean for each genotype (N=3 mice for each). *p<0.05 as determined by a two-tailed, unpaired T-test. The raw data and specific p-values for the comparisons are presented in Figure 1—figure supplement 4—source data 1.