Supplementary Figure 1: Relative distribution of RE⁺, CB⁺ and GABAergic neurons in LIIa and LIIb.

(a) WFS1 is co-expressed in CB⁺ neurons as shown already previously for the MEC^25,52. Immunohistochemical analysis of WFS1- and CB-expression in the LEC revealed high overlap in excitatory neurons: 93.8 ± 0.5% of all WFS1⁺ neurons in LIIb were CB⁺. Note that no WFS1⁺/GAD⁺ neurons were detected. Thus, immunostaining against WFS1 allows the analysis of excitatory CB⁺ neurons. (b) There is a clear segregation of RE- and WFS1- positivity in LIIa and LIIb, akin to what is observed when employing RE and CB immunohistochemistry. (c) We quantified the number of RE⁺, NeuN⁺ and GAD⁺ neurons in brain slices of GAD-EGFP mice immunostained against RE and NeuN. Excitatory RE⁺ neurons represent almost all excitatory neurons in LIIa, but only a small fraction of excitatory neurons in LIIb. The six panels on the right show higher magnifications of the boxed area. (d) Same as in c, but slices were immunostained against WFS1 and NeuN. WFS1⁺ neurons represent the vast majority of all excitatory neurons in LIIb, but only a very small population of excitatory neurons in LIIa. The six panels on the right show higher magnifications of the boxed area. (e) Table summarizing the relative distribution of RE⁺, WFS1⁺ and GAD⁺ neurons in LEC LII. For each data point, the quantification was performed in 4 slices from 3 hemispheres. The ‘neuron total’ for the data point regarding the quantification of GABAergic neurons includes all NeuN⁺ neurons plus a minority of GABAergic neurons that were EGFP⁺ but NeuN^- (it is known that some GABAergic neurons do not express NeuN). Scale bars in a to d, 50 μm and 10 μm (magnified images). Abbreviations: EGFP, enhanced green fluorescent protein; exc., excitatory; GAD⁺ neuron, GABAergic neuron; L, layer; NeuN, neuronal nuclei; WFS1, Wolfram syndrome 1.