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Cerebellar proteoglycans regulate sonic hedgehog responses during development

¹ Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
² Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA

View larger version (39K): [in a new window]   Fig. 1. Sonic hedgehog contains a Cardin-Weintraub consensus sequence for heparin binding. (A) Alignment of vertebrate sonic hedgehog and Drosophila Hedgehog protein sequences. Identical amino acids are in yellow and the putative heparin-binding domain is in blue. Green highlights indicate two absolutely conserved basic amino acid positions. Pictured below the sequence alignment is the Cardin-Weintraub consensus sequence for heparin binding; B represents basic amino acids. Red asterisk identifies N50, an amino acid involved in binding to PTCH. (B) Sequences of mutant SHH:AP generated in these studies. (C) Molecular model of SHH demonstrating the Cardin-Weintraub sequence (blue), and N50 and S156 (red asterisks), two amino acids involved in binding to patched [adapted from Pepinsky (Pepinsky et al., 2000)].

View larger version (21K): [in a new window]   Fig. 2. Sonic hedgehog interacts with heparin through the Cardin-Weintraub sequence. (A) Elution profile of SHH:AP and AlaSHH:AP from a heparin-agarose column in a continuous salt gradient. SHH:AP and AlaSHH:AP content of each fraction was quantified by its alkaline phosphatase activity. Two peaks are evident for SHH:AP but only one for AlaSHH:AP. (B) Elution behavior of SHH:AP and three mutants. The molarity of peak ligand elution was calculated from curve fits to the plot of NaCl concentration as a function of fraction number. Data are the mean±s.e.m. of three separate experiments. SHH:AP and Arg+SHH:AP behave identically with peaks of elution at 0.5 and 0.75 M NaCl. By contrast, AlaSHH:AP and GlnSHH:AP each display only a single peak of elution at 0.5 M.

View larger version (57K): [in a new window]   Fig. 3. Ext1 and Ext2 expression during early postnatal development in mouse brain. (A) In situ hybridization for Ext1 in a postnatal day 8 mouse brain. Ext1 mRNA is most abundant in the cerebellum. Scale bar: 1 mm. (B) Ext1 and Ext2 have similar patterns of expression in postnatal day 8 mouse cerebellum. Scale bar: 1 mm. (C) Higher magnification views of the cerebellum reveal that both Ext1 and Ext2 are expressed by granule cells of the EGL and IGL as well as by Purkinje cells. E, EGL; M, molecular layer; P, Purkinje cell layer; I,IGL. Scale bar: 25 µm.

View larger version (37K): [in a new window]   Fig. 4. Ext1 and Ext2 expression increases during postnatal cerebellar development. (A) Northern blot analysis of Ext2 expression in developing and adult mouse cerebellum reveals that expression increases with increasing postnatal age, particularly from P4 to P9. Ext2 mRNA is present as two transcripts of 3.5 and 4 kb. (B) Ext1 expression demonstrates a similar increase between P4 and P9. Ext1 mRNA is present as a single species of 2.7 kb. Ethidium Bromide images corresponding to each northern blot are shown as loading controls (numbers refer to postnatal age; A, adult).

View larger version (51K): [in a new window]   Fig. 5. SHH binds to endogenous HSPGs through the Cardin-Weintraub sequence. The binding of SHH:AP and AlaSHH:AP to endogenous HSPGs in postnatal day 3 and 6 cerebellum was evaluated by modification of the method of Friedl (Friedl et al., 1997). (A) SHH:AP and AlaSHH:AP exhibit very low levels of binding (red) to sections from P3 mice. DAPI-stained nuclei appear blue. Significant amounts of SHH:AP but not AlaSHH:AP are bound by P6 HSPGs, suggesting that SHH:AP requires a wild-type Cardin-Weintraub sequence for this binding. Scale bar: 10 µm. (B) Heparinase I and III treatment results in significantly decreased binding of SHH:AP to the EGL and IGL in sections of P6 cerebellum. Scale bar: 10 µm. (C) Higher magnification view of the SHH:AP localization. Mutation of the Cardin-Weintraub sequence (AlaSHH:AP) reduces the total level of binding. Scale bar: 10 µm.

View larger version (21K): [in a new window]   Fig. 6. Interactions between SHH and HSPGs modulate proliferation of more mature granule cells. (A) Proliferation dose response of primary cerebellar cultures from P6 mice to SHH:AP (white squares) or AlaSHH:AP (black diamonds). In this and the following panels, a representative experiment is shown in which data from quadruplicate cultures was averaged and normalized to the mean incorporation observed in the absence of SHH. Values are represented as fold proliferation relative to this control±s.e.m. (B) P6 cultures were treated with SHH in the absence (white squares) or presence (black diamonds) of a mixture of heparinase I and III. In both A and B, P6 cultures display a bell-shaped dose-response to SHH, and disruption of the interaction of SHH and HSPGs reduces the peak proliferative response. (C) Proliferation dose response of primary cerebellar cultures derived from postnatal day 3 (P3) mice to SHH:AP (open squares) or AlaSHH:AP (filled diamonds). (D) P3 cultures were treated with unconjugated SHH in the absence (white squares) or presence (black diamonds) of a mixture of heparinase I and III. In both C,D, P3 cultures display increasing proliferation in response to increasing doses of SHH and disruption of the interaction between SHH and HSPGs has no effect on the proliferative response.

View larger version (14K): [in a new window]   Fig. 7. Mutation of the Cardin-Weintraub sequence and disruption of SHH-HSPG interactions does not alter receptor binding. (A) Fixed cultures were incubated with 1 nM of SHH:AP and increasing amount of unconjugated SHH (5-500 nM). Data are the means of triplicate determinations±s.e.m. Linear fits to the steepest region of the bound/free versus bound curve are given beneath each plot along with calculated values for Kd and Bmax. (B) Cells were incubated for 2 hours on ice with increasing doses of SHH:AP or AlaSHH:AP (0.7 to 35 nM) in the absence or presence of 100 nM unconjugated SHH. Specific binding was derived from the difference between triplicate determinations of binding in the absence and presence of excess unconjugated SHH. Specific binding is plotted as net absorbance in OD units at 405 nm. (C) P6 cultures were treated with a mixture of 1 mU/ml heparinase I and III or not as indicated. Specific SHH:AP binding was measured by incubation of 1 nM ligand in the absence (white bars) and presence (black bars) of 100 nM unconjugated SHH. Data are the means of quadruplicate determinations±s.e.m. and are plotted as the fraction of binding measured for SHH:AP alone.