Figure 2
Developmental downregulation of Gfrα1-TLZ and HB9::GFP expression. (A) Single optical plane confocal image through lamina IX at P5 showing Gfrα1-TLZ expression (A1, Cy3, red), Hb9::GFP (A2, green), ChAT-immunoreactivity (A3, Cy5, blue) and merged images (A4). Most ChAT+ MNs express both markers at P5, but some express strong Gfrα1-TLZ and no Hb9::GFP (white arrowheads). A few large MNs express Gfrα1-TLZ weakly (orange arrowheads), while small Hb9::GFP interneurons (ChAT-, asterisks in A-1-4) do not express Gfrα1-TLZ. (B) Size distribution of P5 ChAT+ (gray bars) and Gfrα1-TLZ+ MNs (white bars); 83.6% of ChAT+ MNs express Gfrα1-TLZ. P5 ChAT+ MNs have small/medium sizes that can be fitted by two overlapping distributions (solid lines) suggesting initial differentiation of small (red line) and large (blue line) populations. Gfrα1+ MNs (dashed lines) are fitted by a similar bimodal distribution. (C) Size distribution of P5 Gfrα1-TLZ+ and Hb9::GFP- MNs (white bars). These cells represent 28.2% of all ChAT+ MNs (gray bars) and are concentrated in small size bins. (D) Similar image series as in A, but at P60 (images are at lower magnification and four optical planes were superimposed to adjust for neuropil spread with age). Gfrα1-TLZ+/Hb9::GFP- MNs (white arrowheads) are quite distinct at this age. Gfrα1-TLZ is largely absent from large MNs and Hb9::GFP+ MNs (orange arrowheads) and many large MNs also lack Hb9::GFP (blue arrowheads). (E) P60 size distributions (as in B). Only 54.1% of ChAT+ cells express Gfrα1-TLZ, with the strongest reduction in the large population. No significant downregulation of Gfrα1-TLZ expression occurs in small ChAT+ cells. (F) At P60, 43.9% of ChAT+ MNs are Gfrα1-TLZ+ and Hb9::GFP-. Their size distribution suggests many large Gfrα1-TLZ+ MNs have lost HB9::GFP. Error bars indicate SEM; 50 μm2 bin size. At P5 average histograms from three animals, while at P60 two animals were averaged (six ventral horns analyzed per animal; 608 ± 7 and 337 ± 7 MNs analyzed per animal at P5 and P60 respectively). Scale bars: (A, B) 100 μm.