Extended Data Fig. 5: Identification of pontine mossy fiber neurons and lower rhombic lip precursors, and analysis of WNT medulloblastoma scRNA-seq.

a, Mossy fiber neuron cluster (n = 198 cells) highlighted in the t-SNE embedding of the P0 mouse pons. b, Left: expression of Olig3, a molecular marker of the lower rhombic lip (LRL), the progenitor ___domain that gives rise to pre-cerebellar neuron populations including mossy fiber (MF) and climbing fiber (CF) neurons^12,71,72. Right: expression of Atoh1, which identifies the MF lineage in the LRL^72,73, and is required for their development⁷⁴. c, Violin plots quantifying expression of genes used to determine cluster identity in the MF neuron population (n = 198 cells). Left: Pax6, Zic1 and Olig3, markers of LRL progenitors that give rise to MF neurons, identified by lineage tracing and loss of function experiments^12,72,73,75. Pax6 regulates cell fate allocation in the LRL⁷³, and Zic1 regulates MF neuron positioning and projections in the developing pons⁷⁵. Middle: Atoh1, a marker of MF lineage in the LRL^72,73,74. Pcsk9, a marker of the pontine nucleus, a prominent structure formed exclusively by MF neurons⁷⁶. Barhl1 is required for the formation of MF nuclei, and is expressed in RL-derivatives except for the inferior olivary nucleus (ION, the structure formed by CF neurons, and the source of climbing fibers to the cerebellum)⁷⁷. Right: Genes marking the climbing fiber neuron lineage⁷², which also originates from LRL precursors, are absent in the MF population, resolving the cluster identity (Ptf1a, Neurog1/Ngn1 and Ascl1/Mash1). Foxd3 is a marker of the mature ION⁷⁸. Brn3a, which marks the ION throughout its maturation⁷⁸, is undetected. Violin plots are generated as in Fig. 7. d-e, PCA of re-clustered pontine progenitors as in Fig. 2a, with cluster containing LRL precursors highlighted (d) (n = 393 cells), or cells colored by expression of selected gene markers for LRL precursors (e). Expression of each gene was normalized to a [0, 1] scale for visualization. f, In situ hybridization of selected markers in the E13.5 mouse from the Allen Brain Atlas illustrating expression patterns in the LRL. g, Expression of ZIC1, CTNNB1 and OTX2, mossy fiber neuron marker genes (BARHL1, PCSK9), and climbing fiber neuron marker genes (BRN3A, ASCL1) in the t-SNE embedding of the WNT-MB-1 patient tumor sample (n = 3,975 cells). Expression of each gene was normalized to a [0, 1] scale for visualization. Similar expression patterns were observed in the other two patient samples. h, Inferred pseudotime reconstruction from the malignant cells, represented in the t-SNE embedding of the WNT-MB-1 patient tumor sample. i-j, Characterization of two patient WNT medulloblastoma scRNA-seq samples as in Fig. 4. Top left: t-SNE and clustering, with non-malignant clusters labeled, and number of cells indicated in parentheses. Top right: expression of marker genes of malignant tumor clusters. Bottom: cells in malignant tumor clusters colored by pseudotime inferred through trajectory analysis.