Figure 4

Dynamic expression of Lrrn1 in the diencepahlon. (a) Wholemount or (b-m) flatmounted specimens subject to double in situ hybridization for Lrrn1 (blue staining) and Fgf8 (a), Shh (b, i, k, l) or Lfng (c-h, j), or Lfng/Shh (m). Colour coded labels in the bottom right of each panel indicate the probes used. (a) Lrrn1 does not overlap with Fgf8 at the MHB (black arrowhead), or with Shh expression in the floorplate in (b). (c) Double in situ hybridization with Lfng shows downregulation in the prZLI region of the diencephalon (arrow) from HH12. (d) A hemisected, flatmounted preparation showing downregulation of Lfng and Lrrn1 in the diencephalic anlage. Lrrn1 is expressed within a wedge-shaped region in which Lfng not expressed (black arrow; boundaries of Lfng expression domains flanking the prZLI territory are marked by red arrowheads). (e) Lrrn1 expression clearly fills the Lfng-free wedge and abuts or partially overlaps the border of Lfng expression caudally. Note the low-level or absence of Lrrn1 expression in the thalamus itself (asterisk). (f) By HH15 the interface of the Lrrn1/Lfng domains has sharpened considerably to reveal the position of the ZLI (marked by black arrowheads) and the diencephalic basal-alar plate boundary (yellow arrowheads). (g) Lrrn1 downregulated in the ZLI at HH17. (h) Higher magnification view of the same specimen showing the ZLI as a Lrrn1/Lfng-free domain (black arrowhead). (i) Double in situ with Lrrn1 and Shh (red staining) showing strong domain of Lrrn1 expression immediately anterior to the ZLI (black arrowheads). (j) Strong expression on the anterior side of the ZLI compartment and the DMB at HH21 (black arrows). (k) At HH23 Lrrn1 abuts the expression domain of Shh on both sides of the ZLI (black arrow) as seen at higher magnification (l). It is expressed in the dorsal prethalamus, although Lfng is not (m). Scale bars = 200 μm.