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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000729

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Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 4114 MRB III, Nashville, TN 37232, USA.

View larger version (72K): [in this window] [in a new window]   Fig. 1. ShhN/- mutant forebrain displays HPE-like phenotypes. (A-F) Frontal view of control (A,B), ShhN/- (C,D) and Shh-/- (E,F) mouse forebrains at E10.5. B,D,F are Hematoxylin and Eosin-stained coronal sections. ShhN/- telencephalon shows enlarged forebrain ventricles (A,C, arrowheads) and lacks MGE bulge (B,D, arrowheads). Additionally, the frontal nasal process (fnp) is closely positioned in the ShhN/- mutant (compare arrows in A and C) and displays a single nasal pit (npt, arrows in B,D). Note that the ventricles of the ShhN/- telencephalon are distinctly different from those of Shh-/-, which are collapsed into a small ventricle (asterisk in F,L). (G-L) Sagittal view of control (G,H), ShhN/- (I,J) and Shh-/- (K,L) embryos at E12.5. H,J,L are Hematoxylin and Eosin-stained sections. Enlarged forebrain (fb), midbrain (mb) and hindbrain (hb) ventricles in ShhN/- are clearly evident (G,I). In addition to MGE, other morphologically distinct structures such as LGE and the choroid plexus (cp) are absent or defective in the ShhN/- telecephalon (arrows and arrowheads in H,J). (M-R) E18.5 control and ShhN/- embryos showing frontal (M,P), coronal (N,Q) and dorsal (O,R) views. Note hypoplasia of the lower jaw (P, arrow), single nostril (P, arrowhead), absence of olfactory bulb (R, arrow) and enlarged cerebral cortex (ctx) and midbrain (R) in ShhN/- mutants. MGE, medial ganglionic eminence; HP, hippocampus primordium; LGE, lateral ganglionic eminence; te, telencephalon; St, striatum.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Cell proliferation, apoptosis and differentiation in the ShhN/- telencephalon. (A-F) TUNEL analysis of sections from E11.5 wild-type (A-C) and ShhN/- (D-F) mouse embryos at the level of ventral (A,D), dorsal (B,E) and dorsal midline (C,F). (G) Bar chart showing that there are more apoptotic cells in ventral and dorsal regions of the ShhN/- telencephalon (D,E, arrows). The number of apoptotic cells in the dorsal midline is comparable between ShhN/- and control (C,F, arrowheads). (H-M) Coronal sections of E11.5 control (H-J) and ShhN/- (K-M) telencephalon labeled with BrdU antibody. (N) Bar chart showing that there is a statistically significant increase in the percentage of BrdU-positive cells in ventral (v), dorsal (d) and dorsal midline (dm) of ShhN/- telencephalic neuroepithelium as compared with controls. (O-X) Immunofluorescence of E11.5 control (O-S) and ShhN/- (T-X) telencephalic sections labeled with Tuj (O-Q,T-V) and Isl1 (R,S,W,X) antibodies to highlight neuronal differentiation. Note that differentiation in the ventral region is visibly reduced in ShhN/- neuroepithelium (compare O,R,S with T,W,X). v, Mash1+ ventral region; d, Pax6+ dorsal region; dm, dorsal midline.

View larger version (73K): [in this window] [in a new window]   Fig. 3. Absence of MGE progenitors in the mouse ShhN/- telencephalon. (A-D) Nkx2.1 expression in control (A), ShhN/- (B), Shhflox/- (C) and Shh-/- (D) telencephalons. Nkx2.1 is an MGE marker (arrow in A points to the boundary of the Nkx2.1 expression domain) and its expression is maintained in Shhflox/-, but lost in ShhN/- and Shh-/-, telencephalons. (E-H) Mash1 expression in control (E), ShhN/- (F), Shhflox/- (G) and Shh-/- (H) telencephalons. Mash1 expression delineates the ventral region of the control telencephalon, encompassing the MGE and LGE. Mash1 expression was reduced in the LGE domain of Shhflox/- as compared with the control. Mash1 expression was maintained in the ventral ShhN/- telencephalon (arrows in E and F point to the boundary of the Mash1 expression domain). No Mash1-positive cells were found in Shh-/- telencephalon at this stage. (I-L) Pax6 expression in the telencephalon of control (I), ShhN/- (J) Shhflox/- (K) and Shh-/- (L) embryos. Pax6 normally marks the dorsal telencephalon with the exception of the distal dorsal midline. Note that weak Pax6 expression extends to the ventral region in the ShhN/- telencephalon (arrows in I and J point to the presumptive boundary between neocortex and LGE). Shhflox/- showed comparable Pax6 expression to that of the control, whereas the Shh-/- telencephalon was completely dorsalized at this stage. Brackets indicate LGE domains. ncx, neocortex; MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Global appearance of LGE fate in ShhN/- telencephalic neuroepithelium at later developmental stages. (A-D) E13.5 control (A-B) and ShhN/- (C-D) mouse coronal sections immunostained with LGE marker, Gsh2 or Mash1 as indicated. Aa and Ca are higher magnifications of the boxed regions in A and C, respectively. (E-Hc) E15.5 control (E-F) and ShhN/- (G-Gc,H-Hc) coronal sections stained with Gsh2 or Mash1 antibody as indicated. Ea-c, Ga-c and Ha-c are higher magnifications of the boxed regions in E, G and H. At E13.5, ectopic Gsh2 or Mash1 expression in ShhN/- is restricted to the dorsal midline (Ca,D). By E15.5, ectopic LGE marker expression was also detected in the neocortex region (Gb,Hb, arrows). (I-Ja) Gli3 expression in E13.5 control and ShhN/- telencephalon. (I,Ia) Strong Gli3 expression was detected in control telencephalic cortex and LGE, weak expression was present in MGE, and no Gli3 expression was detected in cortical hem and developing choroid plexus. (J,Ja) Gli3 expression was prominent along the dorsoventral axis of the ShhN/- telencephalon, but selectively absent in the dorsal midline. MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; ncx, neocortex; st, striatum; tCP, telencephalic choroid plexus.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Ectopic activation of Shh signaling in the dorsal region of the ShhN/- telencephalon. (A,B) E14.5 Ptch1-lacZ (A) and ShhN/-;Ptch1-lacZ mouse coronal sections stained with X-Gal to highlight sites of Shh signaling in the telencephalon. In control embryos, lacZ expresssion is restricted to the basal ganglions, with weaker lacZ expression in the LGE domain (A, arrow). In ShhN/- embryos, weak lacZ expression is additionally detected at many sites in the dorsal telencephalon (B, arrowheads), consistent with ectopic LGE marker expression. (C) Immunoblotting of whole-brain protein extracts followed by incubation with a Gli3-specific antibody recognizing full-length (Gli3-190) and repressor forms (Gli3R) of Gli3. The Gli3R/Gli3-190 ratio is reduced in the E15.5 ShhN/- brain. (D) Bar chart showing Gli3R/Gli3-190 ratio.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Dorsoventral patterning is largely unperturbed in ShhN/- spinal cord. (A-L) Cross-sections of E10.5 control (A,C,E,G,I,K) and ShhN/- (B,D,F,H,J,L) mouse spinal cord stained with the indicated markers of neural progenitors and differentiated neurons at the branchial level. Nkx6.1 is expressed in the broad ventral domains encompassing the p3, pMN and p2 progenitor domains. Olig2 is selectively expressed in the pMN progenitor domain. Pax7 is expressed in all dorsal progenitor domains. The domain that is negative for Pax7 and Nkx6.1 represents the p0 and p1 progenitor domains (brackets in A,B). (M) Quantification of Pax7, Nkx6.1 and p0+p1 progenitor domain size as a percentage of total dorsoventral (D-V) length of the neural tube. (N,O) Quantification of total numbers of cells in the FP (Foxa2+), pMN (Olig2+), p3 (Nkx2.2+), MN (Isl1+), V2 (Chx10+) and V1 (En1+) domains in the wild type and ShhN/-.

View larger version (123K): [in this window] [in a new window]   Fig. 7. Expression of Shh RNA, protein and target genes in ShhN/- neural tube. (A-H) Comparison of Shh RNA and protein expression in control and ShhN/- mouse forebrains. At the nine-somite stage, Shh expression can be detected in the forebrain neuroepithelium (A,E, arrowheads) and the underlying dorsal foregut endoderm (arrows). Note that Shh RNA expression is selectively lost in ShhN/- telencephalon (E,F, arrowheads). Similarly, Shh protein is also undetectable in the ventral ShhN/- telencephalon (H, arrowhead in G), and protein expression in the dorsal foregut is significantly reduced at the eight-somite stage (G, arrow). The dotted line (B,F) encloses optic vesicles. (I,J,M,N) Nkx2.1 and Ptch1 expression in control and ShhN/- forebrain at E9.5. The dotted line encloses optic vesicles. (K,L,O,P) Comparison of expression of Shh RNA and protein in control and ShhN/- spinal cord at E10.5. Shh protein is detected in both the basal and apical surfaces of the floorplate as well as in ependymal cells that line the central canal of the neural tube (L, arrows). However, in ShhN/- embryos, Shh protein resides exclusively at the apical compartment of floorplate cells (arrowheads in P) and does not accumulate at the ependymal cell surface. n, notochord.

View larger version (66K): [in this window] [in a new window]   Fig. 8. ShhN rescues patterning defects in the spinal cord but not in the telencephalon of Disp1-/- mouse embryos. (A-E) The Disp1-/-;ShhN/+ mutant telencephalon shows characteristics similar to those of the ShhN/- telencephalon, including absence of Shh (A) and Nkx2.1 (E) expression, enlarged forebrain ventricles (B, the region enclosed by the dotted line), reduced eye (B, arrow), ventrally extended Pax6 expression (C) and presence of Mash 1 expression (D). Arrows in C and D mark the presumptive boundary between LGE and cortex. (F) Robust Shh protein signal is visualized in notochord and floorplate. Note the absence of Shh protein staining in ependymal cells. (G-J) Dorsoventral patterning is established in Disp1-/-;ShhN/+ mutant spinal cord, as demonstrated by defined expression domains of Pax7 and Nkx6.1 (G). Note the presence of V3 interneuron as shown by Nkx2.2 staining (I) and the expanded pool of floorplate cells (H).