Fig. 1. Trochlear motor neurons are located both within and posterior to the isthmic organiser. (A-D) Chick hindbrains stained for Isl1/2 (brown) and Fgf8 (blue) by combined immunohistochemistry and in situ hybridisation. All preparations are `open-book' flat mounts i.e. cut along the dorsal midline and opened such that the floor plate is medial in the preparation and dorsal regions are lateral. (A) HH25 chick embryo showing the position of trochlear motor nuclei within the anterior hindbrain. Cell bodies are detected ventrally in rostral r1, both within and adjacent to the Fgf8 expression domain. They appear more scattered than oculomotor neurons in the midbrain and most of r1 is essentially devoid of Isl1/2+ motor neurons. (B) The first trochlear motor neurons are detected at HH17 within and ventral to the Fgf8 expression domain. (C) At HH19 trochlear cell bodies are detected both within and posterior to the isthmus. (D) By HH25 the trochlear nuclei are positioned in a dense cluster in anterior r1 whose anterior extent is coincident with that of Fgf8 expression: anterior boundary of r1. III, oculomotor nucleus; IV, trochlear nucleus; V, trigeminal nucleus; i, isthmus; MB, midbrain; r1, rhombomere 1.

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