1 Program in Neurobiology and Department of Pediatrics, Box 209, Childrens 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
*Author for correspondence (e-mail: j-kohtz{at}northwestern.edu)
Accepted April 8, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sonic hedgehog, Forebrain, Telencephalon, Fatty-acylation, Rat
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Shh is the most intensely studied member of the vertebrate hedgehog family (reviewed by McMahon, 2000 and Ingham, 1998). It is secreted from the notocord, specialized mesodermal tissue that underlies the neural tube and has been shown to induce ventral neurons in the spinal cord (van Straaten et al., 1988; Placzek et al., 1990; Yamada et al., 1991; Placzek et al., 1993). In the anteriormost regions of the neural tube, where the notochord is absent, Shh is expressed in the prechordal plate, as well as the ventral midline of the CNS (Marti et al., 1995b; Shimamura et al., 1995). In patterning the ventral neural tube, Shh has been shown to ventralize neurons along the anterior-posterior (A-P) extent of the neuraxis in accordance with their position (Marti et al., 1995; Ericson et al., 1995; Roelink et al., 1995; Chiang et al., 1996; Shimamura and Rubenstein, 1997; Kohtz et al., 1998). What then determines the A-P specificity of Shh inductions? In the posterior neuraxis, ventral neuronal subtypes are produced in a concentration-dependent manner (Roelink et al., 1995; Ericson et al., 1997). High-level exposure to Shh results in the formation of floorplate cells, whereas lower levels result in motoneuron formation. In more anterior regions of the neuraxis, Shh has been shown to cooperate with Bmp7 or Fgf8 to produce ventral forebrain and midbrain neurons, respectively (Dale et al., 1997; Hynes et al., 1995; Ye et al., 1998; Shimamura and Rubenstein, 1997). Thus, concentration and cooperativity are two mechanisms used by Shh to induce diverse ventral neurons.
We have recently begun to study how Shh influences the formation of the rodent ventral forebrain. These studies suggest that developmentally regulated changes in competence determine the specific response of ventral telencephalic neurons to Shh (Kohtz et al., 1998). In the course of these studies, we consistently observed that only Shh purified from baculovirus-infected insect cells and not Escherichia coli was capable of inducing ventral telencephalic neurons (J. D. K. and G. F., unpublished).
Pepinsky et al. (Pepinsky et al., 1998) have shown that N-terminal fatty-acid modification of the human Shh protein results in a 30-fold enhancement in its ability to convert C3H10T1/2 cells to an osteoblast lineage (Kinto et al., 1997). In these studies, it was found that palmitoylated human Shh, and additional lipid-modified forms such as myristoylated, stearoylated and arachidoylated rat Shh, could be purified from insect cells infected with Shh-containing baculoviruses. The increased activity of these lipid-modified forms suggests that they might be responsible for the difference between insect cell- and E. coli-derived Shh in our assays of telencephalic ventral neural induction.
This study compares the abilities of N-terminal fatty-acylated and non-acylated forms of Shh to ventralize neurons in the rodent forebrain, using both in vivo and in vitro techniques. We provide evidence that N-terminal fatty-acylation significantly enhances the ability of Shh to ventralize early striatal neurons expressing Dlx-containing homeobox genes, Mash1, and/or Islet 1/2. These neurons are similar to those found in both the proliferating and differentiating zones of the E14 rat LGE. We also provide evidence that the N-terminal fatty-acylation of Shh is important in vivo. Recent work has shown that injection of a retrovirus encoding wild-type Shh into the E9.5 mouse forebrain results in the ectopic expression of Dlx2 and severe brain deformities (Gaiano et al., 1999). We now show that Shh containing a point mutation at the site of acylation is unable to induce the ectopic expression of Dlx2 or severe brain deformities. These findings suggest that N-terminal acylation plays an important role in Shh-mediated telencephalic patterning and differentiation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Ectopic expression of wild-type Shh and C24S in mouse embryos
A point mutation in the human Shh cDNA was constructed, converting Cys-24 to Ser (E. A. G., unpublished) (Pepinsky et al., 1998). The full-length wild-type and C24S mutant Shh cDNAs were then subcloned into the retroviral vector pCLE (described in detail by Gaiano et. al. 1999), and co-transfected into 293 cells with a plasmid encoding VSV-G protein. The resulting pseudotyped retroviruses were isolated by high-speed centrifugation and titrated on 3T3 cells. Virus (1 µl) at a titer of 5x108 pfu/ml was injected into the forebrain vesicles of E9.5 mice using ultrasound-guided backscatter microscopy (Olsson et al., 1997). Embryos were harvested at E12.5, cut below the forelimbs for photography or processed for in situ hybridization (Schaeren-Wiemers and Gerfin-Moser, 1993) using digoxigenin-labeled antisense Dlx2 RNAs and alkaline phosphatase staining, as described by Gaiano et al. (Gaiano et al., 1999).
Detection of Shh proteins produced by viral constructs
Full-length wild-type Shh or C24S-Shh virus (5 µl) at a titer of 5x108 pfu/ml was incubated with 3T3 cells grown on 10 mm dishes at 80% confluence. Cells were infected for 1 hour, grown for 48 hours, and lysed in ice-cold 10 mM Tris (pH 7.5), 1% NP40. The cellular debris was centrifuged at 10,000 g, and the supernatant was divided in half. Half the sample was acetone precipitated, resuspended in Laemmli sample buffer, loaded on SDS-PAGE and immunoblotted with anti-Shh antibodies (5E1, DHSB) (Ericson et al., 1996). The other half of the sample was diluted 1:10 in PBS, incubated with rabbit anti-Shh antibodies that had previously been conjugated to protein G Sepharose 4B, washed three times with PBS + 0.1% Triton X-100, resuspended in Laemmli sample buffer, loaded on SDS-PAGE, and immunoblotted with mouse anti-Shh antibodies (5E1) (Ericson et al., 1996). We detected virally produced C24S-Shh protein in immunoprecipitated extracts and wild-type Shh protein in acetone-precipitated extracts. This may be due to solubility differences resulting from the presence of the lipid moieties.
Immunoprecipitation of radiolabeled Shh
C17 cells (Snyder et al., 1992) stably transfected with a Shh-containing plasmid (Liu et al., 1998) and a control cell line were labeled for 4 hours in medium containing either 300 mCi/ml of [9,10-3H]myristate (Dupont NEN, specific activity 16 Ci/mmol) or 300 mCi/ml of [9,10-3H]palmitate (Dupont NEN, specific activity 60 Ci/mmol), or both, as indicated in Fig. 9. Cells were fractionated to yield a soluble fraction and a 125,000 g pellet fraction (Degtyarev et al., 1994). Each fraction was made up to a 1x dilution in the following solubilization/immunoprecipitation buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.8% Triton X-100, 0.2% SDS and 1 mM EDTA. Purified monoclonal antibody against Shh (5E1, Ericson et al., 1996) was preincubated with protein G Sepharose (Pharmacia), washed, and then incubated overnight at 4°C with the culture supernatant, the soluble cytoplasmic fraction or the solubilized pellet fractions. The immunoprecipitates were washed, solubilized and separated by SDS-PAGE. Gels were then treated with Amplify (Amersham/Pharmacia Biotech), dried and exposed to KODAK MR film using a Biomax low-energy intensifying screen for 6 weeks at -80°C for lanes 1-4, 1 week for lanes 5-8, and overnight for lanes 9-14 in Fig. 9. Biorad broad range prestained markers were run alongside for size estimation.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
In order to test whether N-terminal fatty-acylation of Shh affects the induction of rat ventral forebrain neurons, we compared the activities of Shh proteins that were acylated in vitro. N-myristoylated Shh (mShhN) and unmodified Shh (uShhN) were compared for their ability to ventralize telencephalic explants at two specific times during development: E9 and E11. All recombinant proteins (mShhN, uShhN and C24S-ShhN) were made in E. coli from the N-terminal signaling fragment of Shh (spanning amino acids 24-197). The N-terminal fatty-acylated protein mShhN was acylated in vitro (see Materials and Methods). The schematic in Fig. 3 shows the region (in blue) of the E11 telencephalic explant used in the assay for ventral neural induction by these forms of Shh. Fig. 2 shows that at 48 nM (1 µg/ml), mShhN induces neurons expressing Dlx, Mash1 and/or Islet 1/2 (Fig. 2A,B,E,F,I,J,M,N). At the same concentration, uShhN induces very few neurons expressing these genes (Fig. 2C,G,K,O). In order to determine if uShhN can induce striatal neurons at higher concentrations, we incubated E11 explants with different concentrations of mShhN and uShhN. Fig. 3 shows that mShhN is significantly more potent at inducing Dlx (green) and/or Islet 1/2 (red) than uShhN. Induction of Dlx and Islet 1/2 is first seen at 3 nM for mShhN (Fig. 3I), but at 48 nM for uShhN (Fig. 3D). At 48 nM, mShhN (Fig. 3C) exhibits a maximal response in which there is widespread induction of Dlx and/or Islet 1/2. However, even at 3070 nM, the maximal response of explants to uShhN remains restricted to a subregion of the explant (compare Fig. 3A, 3070 nM uShhN with Fig. 3C, 48 nM mShhN). Quantitation and graphic representation of Shh-mediated induction of Dlx and Islet 1/2 in E11 telencephalic explants is shown in Fig. 4. The numbers of Dlx- and Islet 1/2- expressing neurons induced by 3070 nM uShhN are only slightly greater than the numbers obtained with 12 nM mShhN. Thus, N-terminal fatty-acylation can enhance Shh activity up to 200-fold, in good agreement with the 160-fold increase in potency of mShh when assayed on C3H10T1/2 cells (Taylor et al., 2001).
|
Shh containing a point mutation at the site of fatty-acylation is unable induce ectopic Dlx2 expression and severe brain deformities in the mouse telencephalon in vivo
Recent studies show that a point mutation converting the N-terminal cysteine of Shh to a serine (C24S) inhibits palmitoylation of Shh in vitro (Pepinsky et al., 1998). In order to determine whether Shh-mediated induction of Dlx2-expressing ventral telencephalic neurons is also affected by this point mutation, retroviruses producing full-length wild-type Shh and the C24S mutant form (C24S-Shh) were injected into mouse E9.5 telencephalic vesicles. Injections at this early stage in development were guided by ultrasound as described (Olsson et al., 1997; Gaiano et al., 1999). Alkaline phosphatase is dicistronic with Shh in the viral constructs and serves as a marker for virally-infected clusters (Fig. 6B,D,G,I,J). Fig. 6A,C,E shows that retroviruses containing wild-type Shh induce Dlx2 ectopically in regions adjacent to the LGE of the ventral telencephalon, a region where Dlx2 is normally expressed. Ectopic expression of Dlx2 occurred in cells overlapping with Shh-infected cells (Fig. 6A) and in regions adjacent to, or several cell diameters from, Shh-infected cells (Fig. 6C,E). This suggests that Dlx2 can be induced by long-range signaling. In addition, ectopic Dlx2 clusters were found more dorsally in regions of the cortex where severe malformations were detected (Gaiano et al., 1999; and data not shown). 37/42 brains injected with wild-type Shh virus contained severe malformations and distensions in dorsal regions of the forebrain (Fig. 6K,L; Gaiano et al., 1999). In contrast, ectopic Dlx2 clusters were not detected when viruses containing the C24S mutant form of Shh were injected (Fig. 6F,H), despite the fact that large virally infected clusters could be detected by alkaline phosphatase staining (Fig. 6G,I). None of the brains (n=38) injected with C24S virus exhibited dorsal malformations similar to the wild-type phenotype (compare Fig. 6K,L (wild-type Shh) versus Fig. 6M,N (C24S-Shh)). In contrast to its lack of activity in the mouse forebrain, Lee et al. have recently shown that the identical C24S-Shh virus causes deformities in the mouse limb (Lee et al., 2001).
|
C24S-ShhN is equivalent to the unmodified N-terminal fragment of Shh in vitro
In order to rule out the possibility that conversion of the N-terminal cysteine to a serine inactivates Shh activity, we compared the in vitro activities of the unmodified N-terminal fragment of Shh (uShhN) and the C24S mutant form of the N-terminal fragment of Shh (C24S-ShhN). Fig. 7B depicts both proteins: uShhN (4) and C24S-ShhN (5). In the E11 telencephalic explant assay described above (see Fig. 3; Kohtz et al., 1998) uShhN is equivalent to C24S-ShhN at inducing Dlx/Islet 1/2-expressing ventral telencephalic neurons (Fig. 8). Both proteins induce Dlx (green) or Islet 1/2 (red) only at the two highest concentrations tested (Fig. 8, 960 nM and 386 nM). Therefore, the conversion of Cys-24 to Ser results in a protein with activity similar to that expected for a non-acylated form of Shh. It has previously been reported that although C24S-ShhN can act as a dominant negative in converting C3H10T1/2 cells to an osteoblast lineage, uShhN and C24S-ShhN have equivalent activities in the induction of spinal cord motoneurons (Williams et al., 1999). Thus, in two different neuronal assays, conversion of the N-terminal cysteine to a serine does not affect Shh activity.
|
Palmitoylated and myristoylated Shh are membrane bound
In order to verify that neural cells are able to produce an N-terminal fatty-acid-modified form of Shh, immunoprecipitation of Shh from the C17 mouse neural cell line was performed. Fig. 9 shows that wild-type Shh immunoprecipitated from C17 cells (Snyder et al., 1992) that have been stably transfected with a plasmid encoding full-length wild-type Shh (Liu et al., 1998), incorporates radiolabel from either 3H-myristate (lane 5) or 3H-palmitate (lane 7), or a mixture of both (lane 4). In addition, lipid-containing wild-type Shh is membrane tethered, as it was detected in the 125,000 g pellet (lanes 4, 5, 7), but not in the cytoplasmic fraction (lane 2) or in the culture supernatant (lanes 6 and 8). When Shh is labeled with 35S-methionine and 35S-cysteine, the majority of the protein is found in the membrane fraction (lane 12), and not in the cytoplasmic fraction (lane 10). However, low levels of a secreted form can be detected in the medium (lane 14). Shh could not be detected in the C17 control cell line lacking the Shh plasmid (lanes 1, 3, 9, 11, 13). These data, along with experiments by Pepinsky et al. (Pepinsky et al., 1998), indicate that lipid-modified Shh can be detected as membrane-tethered forms. While radiolabel can be incorporated into Shh from either 3H-myristate (lane 5) or 3H-palmitate (lane 7), we cannot determine from these data alone whether the myristate is attached directly or is converted to palmitate prior to attachment.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mShhN induces LGE neurons expressing Dlx, Mash1 and Islet 1/2 in E11 rat telencephalic explants, similar to those found in vivo
Characterization of LGE neurons found in the proliferative and post-mitotic zones of the E14 rat LGE show that they co-express combinations of Dlx, Mash1, and Islet 1/2, but not Nkx2.1. BrdU incorporation studies have previously shown that Dlx2- and Mash1-expressing neurons are proliferating and reside in the VZ/SVZ border (Porteus et al., 1994). Using double labeling, we find that Mash1 and Dlx are expressed in both distinct and overlapping populations of neurons in the VZ and SVZ of the LGE. Islet 1/2 is a LIM homeobox protein that has been found in motoneurons after their last division in the spinal cord VZ (Ericson et al., 1996), and in more differentiated neurons derived from presumptive telencephalic chick explants (Gunhaga et al., 2000). We find that, like Dlx- and Mash1- expressing neurons, Islet 1/2 and Dlx are also expressed in both distinct and overlapping populations of neurons. However, Islet 1/2-expressing neurons are found only in the more differentiated region of the LGE. Double labeling with Mash1 and Islet 1/2 show that these genes are expressed in distinct populations of neurons, consistent with their previously published expression patterns in the forebrain (Guillemot and Joyner 1993; Porteus et. al. 1994; Kohtz et al., 1998). In E11 telencephalic explants, mShhN-induced neurons express Dlx, Mash1 or Islet 1/2, co-express Dlx and Mash1 or Dlx and Islet 1/2, but do not co-express Mash1 and Islet 1/2. These data suggest that mShhN-induced neurons express Dlx, Mash1 and Islet 1/2 in similar combinations to proliferating and differentiated populations found in vivo.
Although we find that mShhN-induced striatal neurons express genes characteristic of early striatal neurons, markers characteristic of mature striatal neurons, such as DARP-32 or parvalbumin are not detected (data not shown). Therefore, we do not know whether these neurons are fated to become striatal projection neurons or interneurons, or both. It will be interesting to determine whether cooperative factors can in fact induce the expression of more mature striatal phenotypes in the telencephalon, or whether these are determined by tissue intrinsic factors.
The role of N-terminal fatty acylated Shh in the developing rodent forebrain
Evidence is presented that N-terminal fatty-acylation of Shh enhances potency 200-fold in the mShhN optimal range in the rat forebrain. This enhancement affects neurons that will populate the two major subdivisions of the basal ganglia, the striatum and globus pallidus. One question raised by these results is whether acylation-dependent enhancement occurs in all Shh-dependent signaling contexts. Williams et al. report that acylated and unacylated Shh have equivalent activities at inducing the differentiation of motoneurons in the chick spinal cord (Williams et al., 1999). Thus, acylation does not simply result in super sonic hedgehog activity in all signaling contexts. If acylation enhances Shh activity in the forebrain, is it possible that it diminishes Shh activity in a different context? Is the forebrain the only region that is influenced by acylated Shh? The equivalent activities of acylated and unacylated Shh in the chick spinal cord raise the question of whether the enhanced activity of Shh is species dependent or position dependent. Lee et al. (2001) recently reported that N-terminal fatty-acylated Shh is differentially required during Drosophila and mouse limb development. Thus, it will be important to further characterize the role of N-terminal fatty-acylation of Shh in different regions of the embryo and in different species.
One important difference between Shh-mediated ventral patterning of the telencephalon and more caudal regions of the neural tube is the absence of the underlying notocord and prechordal plate, which are known sources of Shh caudally. In addition, ventral telencephalic neurons are detected before telencephalic Shh expression (Ericson et al., 1995; Fishell, 1997; Shimamura and Rubenstein, 1997; Kohtz et al., 1998). Support for the idea that MGE neurons are specified from early expression of Shh secreted along the anterior primitive streak/Hensens node during gastrulation has recently been reported (Gunhaga et al., 2000). These experiments suggest that there is a gap in timing of exposure to Shh signal and transduction of its signal pathway in the specification of telencephalic neurons. One possibility is that N-terminal fatty-acylation of Shh plays a role in this delay in signal transduction. Experiments to address this possibility are presently being performed.
Fatty-acylation as a mechanism for enhancing the Shh signal
What are the possible effects of fatty-acylation on a signaling molecule such as Shh? Lipid modifications such as myristoylation and palmitoylation have been shown to result in the restriction of proteins to the cell membrane (Goldstein and Brown, 1990; reviewed by Johnson et al., 1994; Casey, 1995). Whereas it is thought that myristoylation is a stable co-translational modification, palmitoylation is dynamic and occurs post-translationally. It has recently been shown that post-translational myristoylation of the pro-apoptotic protein BID targets this protein to mitochondria, thereby enhancing BID-induced release of cytochrome C and cell death (Zha et al., 2000). These authors argue that myristoylation in itself does not target BID to mitochondria, but in fact induces a conformational change in the BID complex. As further support for this hypothesis, the authors cite results from experiments showing that myristoylation of NADH cytochrome b5 reductase is not required for its mitochondrial localization (Borgese et al., 1996). Similar to BID N-terminal myristoylation, N-terminal fatty-acylation of Shh may induce a conformational change in Shh, resulting in a difference in its activity. In addition to membrane localization, acylation is known to affect the activities of proteins in signaling at the cytoplasmic side of the plasma membrane. The presence of lipid moieties on trimeric GTP-binding proteins affects their association and the activities of G-protein coupled receptors (reviewed in Casey, 1995). It has also been found that many of the known acylated proteins contain two lipid moieties. Lipid modifications on Shh are unique in two regards. Experiments by Pepinsky et al. indicate that the N-terminal palmitate on Shh is not thioester-linked, as it is with all other known palmitoylated proteins (Pepinsky et al., 1998). Instead, the palmitate attaches via a stable amide linkage, similar to the attachment of myristoylated proteins. Thus, it remains to be determined whether Shh palmitoylation is dynamic, as it is with other palmitoylated proteins. If Shh palmitoylation is dynamic, it will be important to determine whether specific enzymes modulate the attachment and removal of the palmitate, as have been reported for palmitoylated H-ras (Camp and Hoffman, 1993; Camp et al., 1994). In addition to the N-terminal palmitate, a C-terminal cholesterol modification has been identified on Shh. The C-terminal cholesterol functions in tethering Shh to the cell surface (Porter et al., 1996). It is believed that tethering limits Shh signaling to nearby cells, rendering a short-range signal. Recent evidence indicates that a novel gene, dispatched, may mediate the removal of Shh from the surface of the signaling cell (Burke et al., 1999), thereby allowing Shh to signal in a long-range manner. Evidence provided in this paper suggests that N-terminal acylation may have a role distinct from such tethering.
In our experiments, the use of soluble Shh and explants to assay for Shh activity makes short-range and long-range distance effects indistinguishable. However, if N-terminal fatty-acylation tethers Shh to the receiving cell, increasing its local concentration, then mShhN would exhibit enhanced activities in all the in vitro assays tested compared with uShhN. Given that cells within the explant are exposed to the same concentration of Shh, the ability to detect a difference in the activity of mShhN versus uShhN in rodent ventral forebrain explant assays, but not in chick spinal cord explant assays (Williams et al., 1999; J. D. K. and G. F., unpublished), suggests that N-terminal lipid modification may alter the mechanism of Shh signaling. A number of possibilities may be invoked to explain the enhanced activity of mShhN in rodents. mShhN may bind with higher affinity to its known receptor patched (Marigo et al., 1996; Stone et al., 1996), which has been shown to interact with Shh both biochemically and genetically, or to a different receptor altogether. Another possibility is that the fatty acyl chain may result in rapid internalization of the Shh protein. Outside the cell, N-terminal acylation may prevent the action of inhibitory molecules or overcome a dependence on cooperative factors. The cooperative activities of Shh and Fgf8, Fgf4 and Bmp7 have been described in different Shh signaling contexts (Dale et al., 1997; Hynes et al., 1995; Ye et al., 1998; Shimamura and Rubenstein, 1997). It will be important to determine which of these mechanisms is responsible for the enhancement of N-terminal fatty-acylation in rodent ventral forebrain induction. It should be noted that N-terminal Shh containing two lipid moieties, palmitate at the N terminus and cholesterol at the C terminus, has been isolated (Pepinsky et al., 1998). Thus, the effects of including both lipids on Shh signaling remain to be determined. Further investigation of the effects of fatty-acid modifications on the mechanism of Shh signaling will be essential to our understanding of how ventral telencephalic induction occurs. Moreover, of particular interest will be how these modifications influence Shh activity in a variety of signaling contexts and species.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson S. A., Qui, M., Bulfone, A., Eisenstat, D., Meneses, J., Pedersen, R. and Rubenstein, J. L. (1997). Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons. Neuron 19, 27-37.[Medline]
Anderson K. D. and Reiner A. (1991). Immunohistochemical localization of DARPP-32 in striatal projection neurons and striatal interneurons: implications for the localization of D1-like dopamine receptors on different types of striatal neurons. Brain Res. 568, 235-243.[Medline]
Borgese, N., Aggujaro, D., Carrera, G., Pietrini, G. and Bassetti, M. (1996). A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes. J. Cell Biol. 135, 1501-1513.[Abstract]
Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K-A, Dickson, B. J. and Basler, K. (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803-815.[Medline]
Camp, L. A. and Hofmann, S. L. (1993). Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-ras. J. Biol. Chem. 268, 22 566-22574.
Camp, L. A., Verkruyse, L. A., Afendis, S. J., Slaughter, C. A. and Hofmann, S. L. (1994). Molecular cloning and expression of palmitoly-protein thioesterase. J. Biol. Chem. 269, 23212- 23219.
Casey, P. J. (1995). Protein lipidation in cell signaling. Science 268, 221-225.[Medline]
Casarosa, S., Fode, C. and Guiellemot, F. (1999). MASH-1 regulates neurogenesis in the ventral telencephalon. Development 126, 525-534.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic Hedgehog gene function. Nature 383, 407-413.[Medline]
Corbin, J., Gaiano, N., Machold, R. P., Langston, A. and Fishell, G. (2000). The Gsh-2 homeodomain gene controls multiple aspects of telencephalic development. Development 127, 5007-5020.
Dale, K. J., Vesque, C., Lints, T. J., Sampath, K. T., Furley, A., Dodd, J. and Placzek, M. (1997). Cooperation of BMP-7 and SHH in the induction of forebrain ventral midline cells by prechordal mesoderm. Cell 90, 257-269.[Medline]
Degtyarev, M. Y., Spiegel, A. M. and Jones, T. L. Z. (1994). Palmitoylation of a G protein a1 subunit requires membrane localization not myristoylation. J. Biol. Chem. 269, 30898-30903.
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (1995). Sonic Hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81, 747-756.[Medline]
Ericson, J, Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of long-range Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87, 661-674.[Medline]
Ericson, J., Briscoe, J., Rashbass, P., van Heyningen, V. and Jessell, T. M. (1997). Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube. Cold Spring Harbor Symp. Quant. Biol. LXII, 451-466.
Fishell, G. (1997). Regionalization in the mammalian telencephalon. Curr. Opin. Neurobiol. 7, 62-69.[Medline]
Gaiano, N., Kohtz, J. D., Turnbull, D. H. and Fishell, G. (1999). A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat. Neurosci. 2, 812-819.[Medline]
Goldstein, J. L. and Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature 343, 425-430.[Medline]
Guillemot, F. and Joyner, A. (1993). Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system. Mech. Dev. 42, 171-185.[Medline]
Gunhaga, L., Jessell, T. M. and Edlund, T. (2000). Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo. Development 127, 3283-3293.
Hseih-Li, H. M., Witte, D. P., Szucsik, J. C., Weinstein, M., Li, H. and Potter, S. S. (1995). Gsh-2, a murine homeobox gene expressed in the developing brain. Mech. Dev. 50, 177-186.[Medline]
Hynes, M., Poulsen, K., Tessier-Lavigne, M. and Rosenthal, A. (1995). Induction of midbrain dopaminergic neurons by Sonic Hedgehog. Cell 80, 95-101.[Medline]
Ingham, P. W. (1998). Transducing Hedgehog: the story so far. EMBO J. 17, 3505-3511.
Johnson, D. R., Bhatnagar, R. S., Knoll, L. J. and Gordon, J. I. (1994). Genetic and biochemical studies of protein N-myristoylation. Ann. Rev. Biochem. 63, 869-914.[Medline]
Kinto, N., Iwamoto, M., Enomoto-Iwamoto, M., Noji, S., Ohuchi, H., Yoshioka, H., Kataoka, H., Wada, Y., Yuhao, G., Takahashi, H. E., Yoshiki, S. and Yamaguchi, A. (1997). Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation. FEBS Lett. 404, 319-323.[Medline]
Kohtz, J. D., Baker, D. P. Cortes, G. and Fishell, G. (1998). Regionalization within the mammalian telencephalon is mediated by changes in responsiveness to Shh. Development 125, 5079-5089.
Lazzaro, D., Price, M., De Felice, M. and Di Lauro, R. (1991). The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 113, 1093-1104.[Abstract]
Lee, J. D., Kraus, P., Gaiano, N., Nery, S., Kohtz, J. D., Fishell, G., Loomis, C. A. and Treisman, J. A. (2001). An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev. Biol. (in press).
Liu, A., Joyner, A. and Turnbull, D. H. (1998). Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells. Mech. Dev. 75, 107-115.[Medline]
Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M. and Tabin, C. J. (1996). Biochemical evidence that patched is the Hedgehog receptor. Nature 384, 176-179.[Medline]
Marin, O., Anderson, S. A. and Rubenstein, J. L. (2000). Origin and specification of striatal interneurons. J. Neurosci. 20, 6063-6076.
Marti, E., Takada, R., Bumcrot, D. A., Sasaki, H. and McMahon, A. P. (1995a). Distribution of Sonic Hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537-2547.
Marti, E., Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995b). Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants. Nature 375, 322-325.[Medline]
McMahon, A. P. (2000). More surprises in the Hedgehog signaling pathway. Cell 100, 185-188.[Medline]
Nery, S., Nichterle, H. and Fishell, G. (2001). Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain. Development 128, 1527-1540.
Olsson, M., Campbell, K. and Turbull, D. H. (1997). Specification of mouse telecephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation. Neuron 19, 761-772.[Medline]
Panganiban, G., Sebring, A., Nagy, L. and Carroll, S. (1995). The development of crustacean limbs and the evolution of arthropods. Science 270, 1363-1366.[Abstract]
Pepinsky, R. B., Zeng, C., Wen, D., Rayhorn, P., Baker, D. P., Williams, K. P., Bixler, S. A., Ambrose, C. M., Garber, E. A., Miatkowski, K. et al. (1998). Identification of a palmitic acid-modified form of human sonic hedgehog. J. Biol. Chem. 273, 14037-14045.
Placzek, M., Tessier-Lavigne, M., Yamada, T., Jessell, T. M. and Dodd, J. (1990). Mesodermal control of neural cell identity: floor plate induction by the notochord. Science 250, 985-988.[Medline]
Placzek, M., Jessell, T. M. and Dodd, J. (1993). Induction of floor plate differentiation by contact-dependent, homogenetic signals. Development 117, 205-218.
Porter, J. A., Young, K. E. and Beachy, P. A. (1996). Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255-259.
Porteus, M. H., Bulfone, A., Liu, J. K., Puelles, L., Lo, L., C., Rubenstein, J., L. (1994). DLX-2, MASH-1, MAP-2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain. J. Neurosci. 14, 6370-6383.[Abstract]
Roelink, H., Porter, J. A., Chiang, C.,Tanabe, Y., Chang, D. T., Beachy, P. A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of Sonic Hedgehog autoproteolysis. Cell 81, 445-455.[Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells; in situ hybridization using digoxigenin-labeled cRNA probes. Histochemistry 100, 431-440[Medline]
Shimamura, Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L. (1995). Longitudinal organization of the anterior neural plate and neural tube. Development 121, 3923-3933.
Shimamura, K and Rubenstein, J. L. (1997). Inductive interactions direct early regionalization of the mouse forebrain. Development 124, 2709-2718.
Snyder E. Y., Deitcher, D. L., Walsh, C., Arnold-Aldea, S., Hartwieg, E. A. and Cepko, C. L. (1992). Mutipotential neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68, 33-51.[Medline]
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F. and Rosenthal, A. (1996). The tumor suppressor gene patched encodes a candidate receptor for Sonic Hedgehog. Nature 384, 119-120.[Medline]
Sussel, L., Marin, O., Kimura, S., Rubenstein, J. L. (1999). Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126, 3359-3370.
Szucsik, J. C., Witte, D. P., Li, H., Pixley, S. K., Small, K. M. and Potter, S. S. (1997). Altered forebrain and hindbrain development in mice mutant for the Gsh-2 homeobox gene. Dev. Biol. 191, 230-242.[Medline]
Taylor, F., Wen, D., Garber, E., Carmillo, A. N., Baker, D., Arduini, R. M., Williams, K. P., Weinreb, P. H., Rayhorn, P., Hronowski, X. et al. (2001). Enhanced potency of human Sonic Hedgehog by hydrophobic modification. Biochemistry (in press).
Thor, S., Ericson, J., Brannstrom, T. and Edlund, T. (1991). The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat. Neuron 7, 881-889.[Medline]
Torreson, H., Potter, S. S. and Campbell, K. (2000). Genetic control of dorsal-ventral identity in the telencephalon: opposing roles of Gsh2 and Pax6. Development 127, 4361-4371.
van Straaten, H. M. W., Hekking, J. M. W., Wiertz-Hoessels, E. I., Thors, F. and Drukker, J. (1988). Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo. Anat. Embryol. 177, 317-324.[Medline]
Williams, K. P., Rayhorn, P., Chi-Rosso, G., Garber, E., Strauch, K. L., Horan, G. S., Reilly, J. O., Baker, D. P., Taylor, F. R., Koteliansky, V. and Pepinsky, R. B. (1999). Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity. J. Cell Sci. 112, 4405-4414.
Yamada, T., Placzek, M., Tanaka, H., Dodd, J. and Jessell, T. M. (1991). Control of cell pattern in the developing nervous system: polarizing activity of the floor plate-notocord. Cell 64, 635-647.[Medline]
Ye, Weilan, Shimamura, K., Rubenstein, J. L. R., Hynes, M. A. and Rosenthal, A. (1998). FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93, 755-766.[Medline]
Yun, K., Potter, S. and Rubenstein, J. L. (2001). Gsh2 and Pax6 play complementary roles in dorsal ventral patterning of the mammalian telencephalon. Development 128, 193-205.
Zha, J., Weiler, S., Oh, K. J., Wei, M. C., Korsmeyer, S. J. (2000). Post-translational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 1761-1765.