Fig. 3. The C-terminal lipid diminishes the early striatal neuronal differentiation-inducing activity of N-terminal lipid-containing Shh. (A) Secreted Shh proteins containing different N-and C-terminal lipids were purified by anti-Shh (5E1) affinity chromatography and probed with anti-Shh. (Lane 1) wtShh; (lane 2) C24S-Shh; (lane 3) C24S-ShhN; (lane 4) ShhN; (lane 5) untransfected culture supernatant control from the parental C17 cell line; (lane 6) uShhN (7 ng); (lanes 7,8) overexposures of purified wtShh protein showing slower migrating forms; (lane 9) membrane-extracted wtShh before application to 5E1 affinity column; (lane 10) 0.2 M glycine-eluted protein from 5E1 column; (lane 11) membranes extracted wtShh protein in the flow through after application to the 5E1 column. For lanes 9 and 10, wtShh protein was extracted with 0.1% Triton X-100 from wtShh stably transfected C17 cell membranes. (B) E11 rat telencephalic pieces were dissected from the blue region as indicated. The distances from the midline (red and green lines) were measured using a custom-made grid for the Leica MZ-12.5 microscope, placed in the eyepiece. The proximal cut is 125 µm from the midline, as measured by the green line; the distal cut is 625 µm from the midline, as measured by the red line. The resulting explant (blue) averages 500 µmx375 µm. T, telencephalon; D, diencephalon; M, mesencephalon; A, anterior; P, posterior. (C-N) E11 rat telencephalic explants (n=8) were cultured in the presence of different concentrations (1 nM, 3 nM and 12 nM) of N- and C-terminal lipid-containing proteins and stained for the early striatal neuronal markers Dlx (green) and Islet1/2 (red). wtShh (C,G,K), C24S-Shh (D,H,L), ShhN (E,I,M), C24S-ShhN (F,J,N). No Dlx- or Islet1/2-expressing cells were detected in the absence of Shh (data not shown). (O) Quantitative representation of Dlx induction in early striatal neurons by different N- and C-terminal lipid-containing Shh proteins. Explants shown in C-N, and treated with myristoylated ShhN (mShhN, not shown), were measured for Dlx density (fluorescent intensityxarea of fluorescent signalx10⁶) using Open Lab software. Standard deviation measurements are shown above each bar.