Fig. 5. Morphologically defined MLI types do not align with transcriptionally defined signatures but can be described by marker combinations.

a–c morFISH co-staining of single fluorescently-labeled MLIs with smFISH for t-type markers Sorcs3, Nxph1, Grm8, or Cacna1e. Each panel (i.e. a, a′, a″) shows three MLI soma from separate morFISH co-stainings. a m-type basket cell expresses Sorcs3 but not Nxph1, and expresses high Grm8 and low Cacna1e (a′, a″). b m-type stellate cells with long-range axons express Sorcs3 but not Nxph1, and express Cacna1e and lower Grm8 (b′, b″). c m-type stellate cells with short-range axons express Nxph1 and lower Sorcs3; Grm8 is absent but low levels of Cacna1e are detected (c′, c″). d Violin plot summarizing smFISH puncta quantifications for markers within morphologically defined BCs (left, n = 16 cells), long-range SCs (middle, n = 24 cells), and short-range SCs (right, n = 5 cells). Note the Sorcs3+; Grm8^HIGH; Cacna1e^LOW pattern among the BCs, the Sorcs3+; Grm8^LOW; Cacna1e^HIGH among m-type long SCs, and the exclusion of Grm8 in short-range SCs (c′). e SCs with long-range axons in the upper ML are either Sorcs3+ or Nxph1+. Images and quantifications from a–e are from four animals. f Comparison of MLI transcriptional and morphological identities, as assessed through PAGA. By transcriptional signatures, continuous heterogeneity is present between long-range BCs (orange) and SCs (blue), while short-range SCs (teal) are discretely separated from both transcriptional subtypes. By morphological signatures, there is a discrete division between BCs and SCs, with continuous heterogeneity spanning long-range and short-range SCs. g Schematic summarizes the expression patterns of t-type transcriptional markers among m-type MLIs. Scale bars are 35 µm in a–d; 20 µm in e; 5 µm in inset, a′, a″–c′, c″.