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First published online 4 August 2004
doi: 10.1242/dev.01301

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Synergistic and antagonistic roles of the Sonic hedgehog N- and C-terminal lipids

Jianchi Feng^1,*, Bryan White^1,*,, Oksana V. Tyurina^2,, Burcu Guner², Theresa Larson^1,, Hae Young Lee^1,¶, Rolf O. Karlstrom² and Jhumku D. Kohtz^1,**

¹ Program in Neurobiology and Department of Pediatrics, Children's Memorial Institute for Education and Research and The Feinberg School of Medicine, Northwestern University, Chicago, IL 60614, USA
² Department of Biology, University of Massachusetts, Amherst, MA 01003, USA

View larger version (54K): [in a new window]   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.

View larger version (57K): [in a new window]   Fig. 5. Shh ventralizing activity in the zebrafish forebrain requires the N- or C-terminal lipid. Zebrafish embryos were injected at the two- to four-cell stage with 75 pg of in vitro synthesized mRNA encoding different lipid modifiable forms of Shh. Changes in expression of different Shh responsive genes were assayed at 20 hours by in situ hybridization. (A,D,G,J) nk2.1b (blue) and emx1 (red) double labeling, lateral views of brain, eyes removed. (A,D,G) Expression of different N- and C-terminal lipid-containing forms of Shh leads to variable expansion of nk2.1b into the dorsal telencephalon and the simultaneous reduction of dorsal emx1 expression (arrowheads show the border of nk2.1b and emx1 expression). The effects of injections were categorized as mild, moderate or major, based on the degree of nk2.1b expansion and emx1 reduction (red arrowheads). (J) In uninjected embryos, nk2.1b is expressed in a small band in the ventral telencephalon (red arrow), as well as in the underlying diencephalon. Emx1 expression in the dorsal telencephalon is complementary to that of nk2.1b. (B,E,H,K) nk2.2 expression, lateral views of brain, eyes removed. (B,E,H) Examples of variable expansion of nk2.2 expression (arrowheads indicate the extent of expansion of the nk2.2 expression domain). (K) In uninjected embryos, nk2.2 is expressed in the dorsal diencephalon and in the ventral midbrain, hindbrain and spinal cord (data not shown). (C,F,I,L) pax6 and emx1 expression, dorsal views of the head. (C,F,I) Examples of reduced pax6 (red arrows) and emx1 (white arrows) expression. (L) In uninjected embryos, pax6 is expressed in the eyes, while emx1 expression is seen in the telencephalon between the eyes. (M) Graph showing the percentage of embryos with differently affected gene expression patterns after expression of different Shh N- and C-terminal lipid-containing forms. Gene expression is shown at the top, numbers of embryos assayed in each experiment is shown in each bar. Blue, normal gene expression; yellow, mild ventralization; green, moderate ventralization; red, major ventralization; Shh, wild-type Sonic Hedgehog; ShhN, Shh lacking the C-terminal lipid; C24SShh, Shh lacking the N-terminal lipid; C24S-ShhN; Shh lacking both lipids; di, diencephalon; mb, midbrain; tel, telencephalon.

View larger version (25K): [in a new window]   Fig. 1. Characterization of different Shh proteins in rat E11 brain and limb tissue. (A) Comparison of different N- and C-terminal lipid-containing Shh proteins expressed by stably transfected cell lines and embryonic tissue. The neural cell line C17 was transfected with different Shh constructs in order to obtain stable cell lines expressing different N- and C-terminal lipids. Whole-cell lysates from stably transfected cells were separated by SDS-PAGE and used to compare the migration of Shh proteins present in E11 embryonic tissues. Western blot analysis using anti-Shh (1-10, 11-16) or anti-tubulin (7'-10' antibodies). (Lane 1) uShhN (7 ng; Shh made in E. coli containing no N- or C-terminal lipids). Lanes 2-5 show lysates of C17 cells stably expressing different lipid-containing Shh proteins (described in Table 1). (Lane 2) wtShh (N+C lipid); (lane 3) C24S-Shh (C lipid alone); (lane 4,) C24S-ShhN (no lipid, 10-fold more cell lysate was loaded in order to detect cell-associated protein); (lane 5) ShhN (N lipid alone). (Lane 6) uShhN (5 ng). (Lane 7) Rat E11 midbrain/hindbrain (MB/HB) tissue lysate. (Lane 8) Rat E11 MB/HB lysate diluted 4-fold in denaturing lysis buffer containing 1% SDS. (Lane 9) Rat E11 forebrain (FB) tissue lysate. (Lane 10) Rat E11 limb tissue lysate. (Lanes 7'-10') Lanes 7-10 were stripped and re-probed with anti-tubulin antibody as a loading control. (Lane 11) uShhN (10 ng). (Lane 12) E11 rat MB/HB tissue lysate. (Lane 13) E11 rat FB tissue lysate. (Lane 14) Lysate of C17 cells stably expressing C24S-Shh protein. (Lane 15) Rat E11 embryo tissue lysate with brain, limbs and branchial arches removed. (Lane 16) Rat E11 limb tissue lysate. Lanes 11-16 were exposed for a shorter amount of time. Size standards are shown on the left. The three arrows on the right highlight the migration of different Shh proteins in the range of those detected in C17 transfected lysates. The asterisk marks a form of Shh that is present in the brain and absent from limb tissue. (B) A comparison of the profile of Triton X-100 extractable membrane Shh proteins containing different N- and C-lipids from Shh stably transfected cells, and from embryonic midbrain/hindbrain and limb tissues. Triton X-100 extracted membranes were analyzed by western analysis using anti-Shh antibody. Membranes isolated from Shh stably transfected cell lines (lanes 1-7), E11 rat embryonic midbrain/hindbrain (lanes 8-14) or E11 rat embryonic limb (lanes 16-22) were treated with increasing amounts of Triton X-100: 0.1% (lanes 1,8,16), 0.2% (lanes 2,9,17), 0.4% (lanes 3,10,18), 0.6% (lanes 4,11,19), 0.8% (lanes 5,12,20), 1% (lanes 6,13,21). (Lanes 7,14,22) Pellet; (lanes 15,23) uShhN, 10 ng.

View larger version (27K): [in a new window]   Fig. 2. The N and C-terminal lipids act synergistically to restrict Shh to the membrane. (A) Anti-Shh western analysis of lysates derived from C17 cells expressing different lipid-containing Shh proteins. M, membrane bound (whole cell lysates); S, secreted (cell culture supernatants immunoprecipitated using 5E1 monoclonal antibody). (Lane 1) uShhN (7 ng; Shh made in E. coli containing no N- or C-terminal lipids); (lanes 2,3) C17 (non-transfected parental cell line); (lanes 4,5) C24S-Shh (C lipid alone); (lanes 6,7) wtShh (N+C lipid); (lanes 8,9) C24S-ShhN (no lipid); (lanes 10,11) ShhN (N lipid alone). Size standards are shown on the left. (B) The C17 neural cell lines expresses Ski1 and mouse dispatched, but not Ski2. cDNA isolated from C17 cells was subjected to PCR using primers against Ski1, Ski2 and dispatched (Disp), as indicated, and loaded on a 2% agarose gel stained with ethidium bromide to visualize PCR products.

View larger version (51K): [in a new window]   Fig. 4. Multimerization is required but not sufficient for Shh early striatal neuronal differentiation-inducing activity. Gel filtration of Shh allows the classification of Shh multimers into three distinct size groups: >669 kDa (L, large), 669-69 kDa (M, medium), and monomers. Different N- and C-terminal lipid-containing proteins from cell culture supernatants derived from C17 cell lines (Fig. 3A) were affinity purified on a 5E1 anti-Shh antibody column. Affinity-purified proteins were separated by Superdex 200 gel filtration column chromatography (AKTA FPLC). Fractions eluted from the column were separated by SDS-PAGE, and probed with anti-Shh antibody by western analysis. (A) wtShh (N+C lipid), (B) C24S-Shh (C lipid alone), (C) ShhN (N lipid alone), (D) C24S-ShhN (no lipid). The elution profile of standard proteins on the Superdex column (Pharmacia) is indicated at the top of each gel. The fraction number is indicated at the bottom of each lane. SDS-PAGE standards are indicated on the left. (E-H) A comparison of the early striatal neuronal differentiation-inducing activities of multimeric wtShh, multimeric C24S-Shh, multimeric ShhN and monomeric ShhN. Rat E11 telencephalic explants were treated with multimeric wtShh (E; fractions 37 and 38; 8/8 explants lacked Dlx- or Islet1/2-expressing cells), multimeric C24S-Shh (F; fractions 37 and 38; 7/8 lacked Dlx- or Islet1/2-expressing cells), multimeric ShhN (G; fractions 37 and 38; 6/7 explants contained Dlx- and Islet1/2-expressing cells) or monomeric ShhN (H; fractions 59 and 61; 6/7 explants lacked Dlx- or Islet1/2-expressing cells). Anti-Dlx was detected with an anti-rabbit Cy2-conjugated antibody (green), and anti-Islet1/2 was detected with an anti-mouse Cy3-conjugated antibody (red).

View larger version (36K): [in a new window]   Fig. 6. Model proposing that the C-lipid mediates long-range signaling of wtShh by facilitating both multimerization and inactivity. Different N- and C-terminal lipid-containing Shh multimers are secreted in the ventral forebrain. The C-terminal lipid-containing multimers exhibit very little differentiation-inducing activity during short-range signaling. During long-range signaling, we propose that the C-lipid enables Shh to form L multimers required for transport, and at the same time maintains Shh in an inactive state, either by rendering it susceptible to an inhibitor (I) or by necessitating activation by an activator (X). One possible mechanism of activation is shown, in which removal of the C-lipid from N+C lipid-containing L and/or M multimers results in the generation of highly active N-lipid M multimers.

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