1. A Hedgehog-Insensitive Form of Patched Provides Evidence for Direct Long-Range Morphogen Activity of Sonic Hedgehog in the Neural Tube 
James Briscoe, Yu Chen, Thomas M. Jessell, Gary Struhl 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1 A Model of Patched and Smo Function in Hh Signal Transduction
(A) Schematic diagram showing the transmembrane proteins Patched (Ptc) and Smoothened (Smo). In the absence of Hedgehog (Hh), Ptc inhibits the Smo-dependent activity of the Hh signal transduction pathway. The second large extracellular loop of Ptc, positioned between transmembrane domains 7 and 8, is depicted in orange.
(B) The binding of Hh to Ptc relieves the inhibition of the Hh pathway and permits Smo to activate downstream targets (blue arrow).
(C) PtcΔloop2 lacks the second large extracellular loop (see Experimental Procedures section) and is not able to bind Hh ligands, but can still inhibit the Hh pathway, even in the presence of Hh protein.
(D and E) A model for the influence of Ptc1Δloop2 on neural patterning. (D) Expression of mPtc1Δloop2 by cells in the ventral neural tube results in a cell-autonomous reduction in Shh signal transduction (indicated as a reduced density of blue dots). As in Drosophila, neural cells expressing mPtc1Δloop2 appear unable to restrict the movement of secreted Shh, resulting in the abnormal dorsal spread of Shh. (E) Schematic diagram showing changes in neuronal cell fate within and dorsal to clusters of cells that express mPtc1Δloop2. mPtc1Δloop2-expressing cells exhibit a ventral-to-dorsal shift in neuronal fate. Just dorsal to mPtc1Δloop2-expressing cells, there is a dorsal-to-ventral shift in neuronal fate. For details, see text
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Clones of PtcΔloop2-Expressing Wing Cells Cannot Sequester or Transduce Hh
(A and B) Wings bearing clones of PtcΔloop2 expressing cells in the anterior (A) or posterior (B) portion of the A compartment. Each wing carries a large A compartment clone (the clone border is outlined in red on the wing, and the clone territory is shown in green in the diagram). Both wings also contain additional clones that populate the P compartment (the border demarcating mutant P compartment cells is outlined in blue and the P compartment is shown in dark blue in the diagram). The red stripe in both diagrams indicates the region formed by A compartment cells that expresses Dpp in response to Hh (yellow arrow) emanating from P compartment cells. In (A), the A compartment clone forms the anterior-most portion of the A compartment and appears wild type. In (B), the A compartment clone forms the posterior portion of the A compartment where it abuts the A/P boundary and is associated with the formation a mirror-symmetric double A winglet as well as the loss of P compartment pattern (discussed in the text). This phenotype is indistinguishable from that associated with similarly positioned clones of smo− cells (Chen and Struhl, 1996). Wings are shown anterior to the left, with longitudinal wing veins 1–5 and posterior wing margin (m) indicated along the wing periphery. PtcΔloop2-expressing cells are marked by the absence of the y+ gene.
(C and D) Wing imaginal discs bearing clones of PtcΔloop2-expressing cells (marked by the coincident expression of GFP) in the P compartment and in either the anterior (C) or posterior (D) portions of the A compartment. The discs are oriented anterior to the left, dorsal up, and the A compartment is marked by the expression of Ci (blue). Cells expressing Dpp in response to Hh are marked by expression of a dpp-lacZ reporter gene (β-gal expression is shown in red). In (C), both the A and P compartments appear to be of normal size, and Dpp is expressed in a stripe of A compartment cells that abuts the A/P compartment boundary, as in wild-type wing discs (the overlap of the red [dpp-lacZ] and blue [Ci] stains appears purple). In (D), a large A compartment clone is located next to the A/P boundary, and all cells belonging to the clone fail to express Dpp, despite the presence of Hh-secreting P compartment cells just across the A/P boundary (the overlap between the green [GFP] stain, which marks PtcΔloop2-expressing cells, and blue [Ci] stain appears turqoise). Immediately anterior to this clone, a stripe of wild-type cells express Dpp (red), indicating that they are responding to Hh secreted by P compartment cells
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Expression of mPtc1Δloop2 in the Chick Neural Tube Inhibits Ptc1 Expression without Affecting Floor Plate Specification
(A) Diagram summarizing in ovo electroporation.
(B–I) HH stage 10–12 embryos electroporated with mPtc1Δloop2 and incubated until HH stage 18 (B and C) or HH stage 22 (D–I). Cells expressing PtcΔloop2 were identified by expression of GFP (green; B, D, F, G, and I). (B and C) Adjacent sections of a transfected neural tube showing expression of mPtc1Δloop2, indicated by GFP expression (B) and the expression of chick Ptc1 (C). (D–F) The expression of HNF3β (red; E and F) is unaffected. GFP (green, C, F) marks cells expressing mPtc1Δloop2. (G–I) Expression of Shh (red; H and I) in neural tube transfected with mPtc1Δloop2 (green, G and I). The results are representative of eight electroporated embryos
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Shh-Mediated Repression of Pax7, In Vitro, Is Inhibited by Expression of mPtc1Δloop2
Expression of Pax7 (red; B, C, E, F, H, I, K, and L) in untransfected [i] explants (A–C and G–I) or mPtc1Δloop2 transfected [i] explants (D–F and J–L) grown in vitro for 24 hr with 1 nM Shh-N (A–F) or 4 nM Shh-N (G–L). Explants expressing mPtc1Δloop2 (green; D, F, J, and L) contain Pax7-expressing cells. Only GFP-containing cells express Pax7 (F and L). The results are representative of 46 explants (8 electroporated and exposed to 1nM Shh-N; 20 electroporated and exposed to 4nM Shh-N; 18 control explants)
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Expression of mPtc1Δloop2 Results in Ectopic Ventral Expression of Pax7 and Pax6
HH stage 10–12 embryos were electroporated in ovo with mPtc1Δloop2 and assayed at stage 24 for expression Pax7 and Pax6.
(A–F) Ectopic expression of Pax7 (red; B, C, E, and F) in the ventral neural tube of embryos expressing mPtc1Δloop2 (green; A, C, D, and F). Not all cells that express mPtc1Δloop2 express Pax7 (arrowhead and see Results section). (F) shows the merged image of the area boxed in (E).
(G–I) The expression of mPtc1Δloop2 (green; G and I) in the p3 domain (bracket, H) ventral to the normal limit of Pax6 expression results in cell-autonomous ectopic expression of Pax6 (red; H and I). The results are representative of 27 electroporated embryos
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
mPtc1Δloop2 Expression Changes the Pattern of Dbx and Nkx Protein Expression
HH stage 10–12 embryos were electroporated in ovo with mPtc1Δloop2 (A–F and M–R) or Pax7 (G–L) and assayed at stage 24 for expression of Dbx1 and Dbx2 (A–L) or Nkx6.1 (M–O) or Nkx2.2 (P–R).
(A–C) Expression of mPtc1Δloop2 (green; A and C) affects expression of Dbx1 (red; B and C). Ventral to the normal limit of Dbx1 expression (dotted lines), mPtc1Δloop2 expression induces ectopic Dbx1 expression in a cell-autonomous manner. Within the normal domain of Dbx1 expression, both dorsal and ventral to the Pax7 boundary, expression of mPtc1Δloop2 inhibits Dbx1 expression in a cell-autonomous manner. Arrowheads mark the approximate ventral limit of Pax7 expression.
(D–F) Expression of mPtc1Δloop2 (green; D and F) ventral to the normal limit of Dbx2 expression (dotted lines) results in the ectopic, cell-autonomous induction of Dbx2 (red; E and F). Within the normal domain of Dbx2 expression, mPtc1Δloop2 inhibits Dbx2. Regions of Dbx2 expression dorsal to the Pax7 boundary are also affected. Arrowheads mark the ventral limit of Pax7 expression.
(G–I) Ectopic expression of Pax7 (green; G and I) does not affect Dbx1 expression (red; H and I).
(J–L) Ectopic expression of Pax7 (green; J and L) does not affect Dbx2 expression (red; K and L). The results are representative of 10 embryos.
(M–O) Ectopic expression of mPtc1Δloop2, identified by GFP expression (green; M and O), inhibits Nkx6.1 expression in neural progenitor cells (red; N and O).
(P–R) Ectopic expression of mPtc1Δloop2 identified by GFP expression (green; P and R) inhibits Nkx2.2 expression (red; Q and R). The results are representative of 15 (Nkx6.1) and 23 (Nkx2.2) electroporated embryos
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 8
A Dorsal-to-Ventral Change in Neural Fate in Cells Dorsal to Clusters of mPtc1Δloop2-Expressing Cell
HH stage 20–24 embryos transfected with mPtc1Δloop2 contained small ventral clusters of transfected cells. These regions of transfected embryos were assayed for Pax6 (A–C) and Nkx2.2 (D–I) expression. (A–C) Pax6 expression (red; B and C) is increased in cells that express mPtc1Δloop2 (green; A and C; arrow). Dorsal to mPtc1Δloop2 cells, the level of Pax6 expression is decreased. (D–I) Nkx2.2 expression (red; E, F, H, and I) is inhibited in mPtc1Δloop2-expressing cells (green; D, F, G, and I; arrow). However, dorsal to the domain of mPtc1Δloop2 expression, cells ectopically express Nkx2.2. The results are representative of 15 electroporated embryos
Molecular Cell 2001 7, DOI: ( /S (01) )

9. Figure 7
An Altered Pattern of Ventral Neurogenesis after Expression of mPtc1Δloop2
(A) Summary of the relationship between progenitor domains and their neuronal progeny.
(B–M) Generation of Evx1/2+ V0 neurons (red; B–D), En1+ V1 neurons (red; E–G), Chx10+ V2 neurons (red; H–J), and MNR2+ MNs (red; K–M) assayed in embryos transfected with mPtc1Δloop2. Embryos were electroporated at HH stage 10–12 and assayed either at HH stage 22–24 (B–J) or HH stage 18 (K–M). Cells transfected with mPtc1Δloop2 were identified by GFP expression (green; B, D, E, G, H, J, K, and M). The normal domains of generation of each cell type are indicated by dotted lines. Expression of mPtc1Δloop2 ventral to the normal domains of generation of V0, V1, and V2 neurons resulted in the ectopic and cell-autonomous generation of these neuronal subtypes. The expression of mPtc1Δloop2 within the domains of progenitors that normally generate each neuronal subtype resulted in the cell-autonomous repression of the generation of each neuronal subtype. n, notochord. The results are representative of 21 electroporated embryos
Molecular Cell 2001 7, DOI: ( /S (01) )