Fig. 3. Mist1^-/- gastric units show increased numbers of neck-to-ZC transitional cells. (A) Neck and base zones of representative single gastric units are depicted (gastric lumen to left); mucous granules of neck cells are green (GSII lectin) and zymogenic granules of ZCs are red (GIF). Note GSII⁺/GIF^Hi cells early in the base zone of the Mist1^-/- mice. (B) MFI of all cells along the neck or ZC differentiation axis in single representative gastric units from each genotype, using anti-Tff2 as a neck cell marker and anti-GIF as a ZC marker, are graphed. The x-axis represents individual cells ordered as a function of distance from the stem cell; i.e. number 1 is the first Tff2⁺ cell in the upper neck zone, and the highest number designates the cell at the bottom of the base zone. Every cell is quantified with regard to both its mucus content (green) and zymogenic granule (red) content. Note the brief transition (cells 10-12) in the normal unit where cells have both mucous and zymogenic granules; otherwise wild-type cells have either exclusively mucous granules (i.e. are neck cells) or exclusively zymogenic granules (i.e. are ZCs in the base). The Mist1^-/- unit (lower graph) has a longer transition zone (cells 9-16), and the cells at the basal side of the transition zone (cells 11-14) have abundant zymogenic granule staining but also retain mucous granules (these are immature ZCs or TZ2 cells corresponding to the GSII⁺/GIF^Hi cells in A and C). Lines through datapoints generated by a curve-generating algorithm based on a running average of `nine nearest neighbor' datapoints. (C) Scatter plots of all cells quantified in all mice, showing GSII (mucus) versus GIF (zymogen) levels. Red boxes show neck-to-ZC transitional cells of the first transition stage (TZ1), with GSII mean neck cell GSII and GIF within 1 s.d. of mean ZC GIF. Fraction of total cells showing this phenotype is in parentheses. Blue box outlines the cluster of GIF^HiGSII⁺ cells ('immature ZCs' or transition stage 2,'TZ2' cells) with GIF significantly above (i.e. 1 s.d.) mean ZC GIF and GSII 10 in Mist1^-/- mice. Similar scatter plots were also generated for other marker combinations (Tff2-GIF and GSII-Pgc; not shown) (D) Fraction of cells with significantly above average (1 s.d.) ZC marker staining and coexpression of above background (>10 MFI) neck cell marker expression (i.e. TZ2 cells). Black column shows mean ±s.d. of the ratio of the fractional representation of TZ2 cells between Mist1^-/- and Mist1^+/- animals. (E) Distribution as a function of cell position within the gastric unit of all cells with ZC marker expression 1 s.d. above mean ZC MFI. Data are the sum across seven mice and 33 gastric units scored per genotype, using the three neck-ZC marker combinations. Cell 0 in this graph defined as the first cell in the gastric unit with ZC expression at or above the ZC mean and neck cell marker MFI <70 (i.e. the first cell at the neck-base interface). Note how Mist1^-/- cells with high ZC marker expression are in early cells (i.e. those nearest the neck), whereas cells with high ZC marker expression tend to occur later in the Mist1^+/- (as the ZCs mature and migrate into the base).