Fig. 5. Shh spread and signaling provides positional information in a graded manner to the neural tube. (A,a) Distinct thresholds of Shh signaling induce differential gene expression indicated by the blocks of color. Changes in the spread of Shh through the neural tube alter DV pattern. (b) Decreasing the sequestration and/or degradation of Shh by blocking the upregulation of the negative regulators Ptch1 and Hhip1 results in an increase in the amount of Shh throughout the neural tube, as compared with the control (dotted line), producing an expansion of the more ventral responses (Jeong and McMahon, 2005); see the schematic of the MtPtch1; Ptch1^-/- mouse embryo ventral neural tube (expressing Ptch1 under the control of the ubiquitous metallothionein promoter, Mt), where the floor plate (FP), p3 and pMN domains expand more dorsally than in a wild-type neural tube. (c) Increasing the diffusivity of Shh, for example by expressing the non-cholesterol-modifiable form of Shh (ShhN), results in an increased range of spread [as proposed by Saha and Schaffer (Saha and Schaffer, 2006)], compared with wild type (dotted line). A consequence of this increased range is decreased Shh accumulation close to the source of secretion and a shallower gradient, which decreases the extent of the highest responses and causes a compaction of ventral neural tube identities (Saha and Schaffer, 2006). (B) The forced, mosaic expression of proteins that inhibit or enhance Shh signal transduction via in ovo electroporation (ectopic expression site highlighted in green) have distinct cell-autonomous and non-autonomous effects on pattern formation in the neural tube. (a) Control electroporation (with GFP alone) has no effect on patterning. (b) The expression of Ptc1^loop2, which does not bind Shh and which inhibits signal transduction, results in a cell-autonomous blockade of signaling (compared with control, dotted line) that inhibits the generation of ventral identities. This also reduces the upregulation of Ptch1 and Hhip1, which would normally sequester Shh protein, thus increasing its spread beyond the cluster of transfected cells. (c) By contrast, the forced expression of Cdo or Gas1, which bind Shh and promote signaling, results in a cell-autonomous enhancement of responses. This increases Shh sequestration, which produces a non-autonomous reduction in the spread of Shh beyond the cluster of transfected cells, which then receive a lower amount of Shh, when compared with control electroporation (dotted line). (C) Shh spreads from ventral to dorsal in the neural tube to establish a gradient. (a) Within the target field, punctae of Shh protein (Shh-GFP; green) are observed apically that accumulate over time in the neural tube; first in volume v1 then in v2 and v3. Shh punctae appear to be associated with the basal bodies (marked by -tubulin, red) of the apically located primary cilia of neuroepithelial cells. (b,c) In neuroepithelial cells abutting the notochord (asterisk), punctae of Shh protein are observed in close proximity to microtubule fibers. The significance of this distribution remains to be determined. Adapted, with permission, from Chamberlain et al. (Chamberlain et al., 2008).