Fig. 5. Local release of Shh from implanted beads is sufficient to inhibit DLHP formation but does not induce an MHP. (A) E9.5 embryos after 3 hours culture following implantation with a single AffiGel blue bead (arrows), adjacent to the neural fold on the right side of the posterior neuropore. The middle embryo has a caudally located bead whereas other embryos have more rostral beads. (B) Sub-division of posterior neuropore into rostrocaudal regions for analysis. (C) Mean ‘asymmetry scores’ of sections containing PBS- or Shh-soaked beads located at each neuropore level. A mean score of 1 (dotted line) indicates symmetrical DHLPs. Scores of 2 and 3 indicate partial and complete suppression of the ipsilateral DLHP respectively. Mean angles of DLHP bending in neural plate with a score of 1, 2 or 3 are shown in Table 1. Mean scores for Shh beads significantly exceed values for PBS beads at rostral and intermediate levels (P<0.001) but not at the caudal position (P>0.05). (D-K) Transverse sections through the posterior neuropore of eight different cultured embryos containing beads (asterisk in D) soaked in PBS (D,F,H,J) or Shh (E,G,I,K). (D,F,H) PBS beads are associated with symmetrical DLHPs in all neuropore regions (27/29 beads in 23/25 embryos) whereas Shh beads regularly suppress formation/maintenance of the ipsilateral DLHP (19/38 beads in 16/31 embryos) in rostral and intermediate neuropore regions (E,G). (I) Complete suppression of DLHP by a caudally located Shh bead. (J,K) Caudal sections of embryos in which a bead has been implanted in the midventral region, beneath the Mode 3 neural plate. There is no sign of MHP formation in embryos containing either PBS (n=2) or Shh beads (n=3). Scale bars: A, 0.5 mm; D-K, 100 µm.
