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N-terminal fatty-acylation of sonic hedgehog enhances the induction of rodent ventral forebrain neurons

¹ Program in Neurobiology and Department of Pediatrics, Box 209, Children’s Memorial Institute for Research and Education, Northwestern University Medical School, 2430 N. Halsted, Chicago, IL 60614, USA
² Developmental Genetics and the Department of Cell Biology, Skirball Institute, New York University School of Medicine, New York, NY 10016, USA
³ Biogen, 14 Cambridge Center, Cambridge, MA 02142, USA

View larger version (102K): [in a new window]   Fig. 1. Ventral telencephalic neurons express Dlx, Mash1, Islet 1/2 and/or Nkx2.1 in distinct and overlapping populations of cells. Coronal sections of rat E14. I (A) Dlx (green), (B) Mash1 (red), and (C-F) Dlx and Mash1 co-expressing cells (yellow). (D-F) Enlargements of the boxed regions in A-C. (D) Proliferative zone of the LGE, double labeled; (E) proliferative zone of the MGE, double labeled; (F) post-mitotic region bordering the LGE and MGE, double labeled. II (A) Dlx (green), (B) Islet 1/2 (red) and (C-F) Dlx and Islet 1/2 co-expressing cells (yellow). (D-F) Enlargements of the boxed regions in A-C. (D) Proliferative zone of the LGE, double labeled; (E) proliferative region of the MGE, double labeled; (F) post-mitotic region of the region bordering the LGE and MGE, double labeled. III (A) Islet 1/2 (green), (B) Mash1 (red) and (C) Islet 1/2 and Mash1 co-expressing cells (yellow, not detected). IV (A) Dlx (green), (B) Nkx2.1 (red) and (C) Dlx and Nkx2.1 co-expressing cells (yellow) – only found in the MGE. (D-F) Enlargements of the boxed regions in A-C. (D) Proliferative zone of the LGE, double labeled; (E) proliferative region of the MGE, double labeled; (F) post-mitotic region of the region bordering the LGE and MGE, double labeled. V Schematic summarizing the protein expression pattern of Shh, Dlx (1,2,5,6), Nkx2.1, Islet 1/2, Dlx 2 and Mash1, based on I-IV, and those previously reported (Kohtz et. al., 1998). L, LGE; M, MGE, white arrow points to the border between the LGE and MGE.

View larger version (111K): [in a new window]   Fig. 2. mShhN specifies neurons in E11 rat telencephalic explants expressing Dlx, Mash1 and/or Islet 1/2, similar to those found in vivo. mShhN (1 µg/ml) -treated explants (A,B,E,F,I,J,M,N), uShhN (1 µg/ml) -treated explants (C,G,K,O), untreated control (-; D,H,L,P). (A-D) Dlx (green), Mash1 (red), and Dlx and Mash1 co-expressing cells (yellow). (E-H) Dlx (green), Nkx2.1 (red). (I-L) Dlx (green), Islet 1/2 (red), and Dlx and Islet 1/2 (yellow), (M-P) Islet 1/2 (green), Mash1 (red), and Islet 1/2 and Mash1 (yellow).

View larger version (43K): [in a new window]   Fig. 3. mShhN is significantly more potent at inducing Dlx and Islet 1/2 in rat E11 telencephalic explants than uShhN. The schematic in the top left-hand corner shows a dorsal view of the E11 rat telencephalon. The blue region delineates the region used in the explant assay for ventral neural induction. The level of induction is indicated by the number of cells expressing Dlx (green), Islet 1/2 (red) or both (yellow). uShhN: A, 3070 nM; B, 384 nM; D, 48 nM; F, 24 nM; H, 12 nM; J, 3 nM. mShhN: C, 48 nM; E, 24 nM; G, 12 nM; I, 3 nM. n=4 for all concentrations except D,E, for which n25. Note that none of the controls treated with buffer alone contained Dlx- or Islet 1/2-positive cells. D, diencephalon; M, mesencephalon; T, telencephalon.

View larger version (25K): [in a new window]   Fig. 4. Quantitative representation comparing the numbers of Dlx- or Islet 1/2-expressing neurons appearing after mShhN or uShhN treatment of E11 rat telencephalic explants. The numbers of cells per 200 µm2 was determined for each concentration of Shh. Four representative fields were counted for each explant; standard deviations were determined using Student’s t-test. Larger deviations at certain concentrations resulted from heterogeneity of the response within the explant (intensely labeled cells adjacent to regions of unresponsive cells).

View larger version (30K): [in a new window]   Fig. 5. mShhN is significantly more potent at inducing Nkx2.1 expression in rat head fold stage (0-4 somites) neural explants than uShhN. The blue region of the schematic shows the region of the headfold used in the explant assay. uShhN: A, 3070 nM; B, 384 nM; D, 48 nM; F, 24 nM; H, 12 nM. mShhN: C, 48 nM; E, 24 nM; G, 12 nM (n=4). Note that none of the controls treated with buffer alone contained Nkx2.1-positive cells (n=6).

View larger version (62K): [in a new window]   Fig. 6. Shh containing a point mutation at the site of N-terminal fatty-acylation does not induce ectopic expression of Dlx2 or severe brain deformities in the mouse telencephalon in vivo. E9.5 mice were injected with retroviruses containing alkaline phosphatase, dicistronic with either full-length wild-type Shh or Shh containing a mutation of the N-terminal fatty acylated residue (Cys-24). Embryos were then harvested at E12.5, coronally sectioned through the telencephalon and processed for in situ hybridization with antisense Dlx2 RNA (A,C,E,F,H) or alkaline phosphatase activity (B,D,G,I,J). Arrows with dotted line indicate ectopic Dlx2 induction (A,C,E); arrows in B,G,I,J indicate virally infected clusters. Sections infected with wild-type Shh virus; (F-I) sections infected with C24S virus. Adjacent sections are as follows: A and B, C and D, E and J, F and G, and H and I. (K,L) An enlarged brain phenotype results in embryos infected with wild-type Shh virus (Shh, n=37/42). Arrows indicate the enlarged region of the forebrain. (M,N) Embryos injected with C24S Shh virus appear normal (C24S, n=38). LGE indicates the location of the lateral ganglionic eminence, where Dlx2 is normally expressed.

View larger version (27K): [in a new window]   Fig. 7. (A) Western analysis of recombinant and virally produced Shh proteins. Recombinant proteins or lysates were loaded onto a 12.5% SDS-PAGE gel, western blotted using 5E1 (anti-Shh antibody) and visualized using a chemiluminescence substrate (NEN). Recombinant proteins (1 µg): 1, C24S-ShhN; 2, mShhN; 3, uShhN; 4 and 5, lysates from virally infected cells (4, C24S-Shh virally infected cell lysate; 5, wild-type Shh virally infected cell lysate. Extract in 4 was immunoprecipitated first and then immunoblotted; extract in 5 was acetone-precipitated first and then immunoblotted. Prestained markers are shown on the left. (B) Schematic of recombinant and virally produced Shh proteins, and their activities.

View larger version (78K): [in a new window]   Fig. 8 The C24S-ShhN protein is equivalent to uShhN in its ability to induce ventral telencephalic neurons expressing Dlx and Islet1/2 in forebrain explants. Rat E11 telencephalic explants were treated with different concentrations of uShhN or C24S-ShhN and stained with anti-Dlx and anti-Islet 1/2 antibodies. The same region of E11 rat telencephalic explant was used for ventral neural induction as in Fig. 3. The level of induction is indicated by the number of cells expressing Dlx (green), Islet 1/2 (red) or both (yellow). Explants were treated with uShhN at the following concentrations: A, 960 nM; C, 386 nM; E, 48 nM. Or with C24S-ShhN: B, 960 nM; D, 386 nM; F, 48 nM (n=4).

View larger version (17K): [in a new window]   Fig. 9. Immunoprecipitation of radiolabeled Shh (arrow) from the mouse neural cell line C17. 3H-palmitate + 3H-myristate incorporation (lanes 1-4). Lane 1: cytoplasmic fraction from control C17 cell line. Lane 2: cytoplasmic fraction from Shh transfected C17 cell line (Shh-C17). Lane 3: 125,000 g pellet, membrane fraction from control C17 cell line. Lane 4: 125,000 g pellet fraction from Shh-C17. 3H-myristate incorporation (lanes 5,6). Lane 5: 125,000 g pellet fraction from Shh-C17. Lane 6: culture supernatant from Shh-C17. 3H-palmitate incorporation (lanes 7,8). Lane 7: 125,000 g pellet fraction from Shh-C17. Lane 8: culture supernatant from Shh-C17. 35S-methionine+cysteine incorporation (lanes 9-14). Lane 9: cytoplasmic fraction from control C17 cell line. Lane 10: cytoplasmic fraction from Shh transfected C17 cell line (Shh-C17). Lane 11: 125,000 g pellet, membrane fraction from control C17 cell line. Lane 12: 125,000 g pellet fraction from Shh-C17. Lane 13: culture supernatant from control C17 cell line. Lane 14: culture supernatant from Shh-C17 (secreted form).