Fig. 8. The OMC and the pRN maintain their anteroposterior relationship, even when the isthmus is shifted rostrally and the midbrain is reduced in size. Rostral movement of the isthmus is induced by mouse Fgf8 misexpression in chick midbrain, which converts midbrain into hindbrain and thereby creates a more rostral midbrain-hindbrain junction. (A) Relationship of the isthmus (IS), identified by WNT1 labeling (blue), to midbrain arc patterning, identified with probes for the homeobox genes PHOX2A (arc 1, blue), PAX6 (P6, brown) and EVX1 (E1, blue). (B) FGF8 gene expression at E5 after unilateral mouse Fgf8 misexpression at E2 documents the rostral shift of the isthmus on the electroporated (right) side, with the left side serving as a control. Asterisk indicates expression of the mouse Fgf8 transgene. Chick FGF8 gene expression, detected by cross-hybridization of the mouse Fgf8 riboprobe, identifies the normal (left) and ectopic (right) isthmi (IS). (C,D) Side views of unelectroporated (C, Control) and Fgf8-electroporated (D, Fgf8 EP) E5 brainstems show that the rostral shift of the isthmus elicits a transformation of the dorsal midbrain from the globose shape characteristic of the tectum into a tubular shape similar to that of the hindbrain. Dorsal is towards the top, and rostral is towards the lower right. Red lines in C and D mark the extent of rhombomere 1. Arrow in D indicates location of the ectopic isthmus induced by Fgf8 misexpression. (E) Elongated trochlear nucleus (PHOX2A+) and compressed midbrain arcs (Hx indicates homeobox genes PHOX2A, PAX6 and EVX1) illustrate the expansion of rhombomere 1 at the expense of ventral midbrain tissue after Fgf8 misexpression. (F) BRN3A gene expression demonstrates rostral shift of the pRN territory after Fgf8 misexpression. Expanded GBX2 labeling demonstrates the concomitant rostral shift in the isthmus (IS) and a transformation of midbrain to hindbrain. (G,H) The PHOX2A+ and BRN3A+ territories of the medial arc maintain their relative anteroposterior positions whether the rostral shift of the isthmus, as identified by WNT1 gene expression (blue in G, brown in H), is moderate (G) or substantial (H). To demonstrate the rostral shift of BRN3A labeling in these wholemounts, the right brainstem has been kinked (G) or the left forebrain has cropped (H). 1, arc 1; III, third ventricle; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus; Tr, trochlear nucleus.