Fig. 3. The pRN originates within the ventral midbrain. Explant cultures of ventral midbrain prepared at E3, a day before a BRN3A+ pRN can be identified, and collected 2-3 days later. (A,B) BRN3A+ pRN is detected in ventral midbrain explants grown in the presence (A) or absence (B) of adjoining dorsal midbrain tissue (PAX3+/BRN3A+). (C,D) Expression of BRN3A (blue) in the ventral midbrain overlaps the rostral half of the PHOX2A (brown) gene expression territory, showing that an organotypic medial arc with appropriate three-dimensional organization is formed in midbrain explants grown with (C) or without (D) tectal tissue. Tectal tissue in cultured midbrain explants is distinguished by two markers: PAX3, which is expressed in dorsal midbrain at the time of culturing; and BRN3A, which is detected in dorsal midbrain by E5. OMC, oculomotor complex; RN, red nucleus; tec, tectum; Tr, trochlear nucleus.