Figure 4.

Overexpression of Sef inhibits auditory nucleus development in chicks. a, a', Bright-field (a) and fluorescent (a') lateral views of an E4 electroporated chick embryo at the level of the otic vesicle (asterisk). Rostral (r) is up and to the right; caudal (c) is down and to the left. eGFP expression can be seen at the level of the otic vesicle and extending rostrally and caudally (arrowheads). 4th v, Fourth ventricle. b, Transverse section through an eGFP-electroporated E4 chick embryo. Strongly labeled cells are present in the rhombic lip (arrowhead) and are also scattered throughout the neural tube, including commissural neurons that have extended axons toward the midline. Only the electroporated side is shown; no labeling is seen on the unelectroporated side. c–f, Coronal sections through control (c, e) and Sef-electroporated (d, f) embryos were hybridized with a probe to cMafb. In control embryos, cMafb is produced in NM medially and in NA laterally. cMafb is also expressed in the nucleus laminaris, which serves a function equivalent to the medial superior olive in mammals. In NA-affected embryos (d), NA is drastically reduced (arrow) on the electroporated side (asterisk), but NM is unaffected. In NM-affected embryos (f), NM is smaller on the electroporated side. Nucleus laminaris (NL) is variably affected, appearing to be shorter and slightly disorganized, but this phenotype was not robust enough to quantify. g, h, The area of NM (in square micrometers) was measured every 28 μm on both sides of the brainstem, moving from rostral to caudal regions (x-axis). Measurements are shown for the embryos depicted in e and f. In the control embryo (g), the area of NM is the same on the unelectroporated (blue) and electroporated (pink) sides. In the Sef-electroporated embryo (h), NM is smaller on the electroporated side in every section. i, Individual (filled symbols) and average (open symbols) NM and NA ratios for control-electroporated (blue) and Sef-electroporated (pink) embryos. The volumes of NM and NA on the electroporated side were compared with the volumes on the unelectroporated side, with a value of 1 predicted as normal. Error bars indicate SD. Although no control-electroporated embryos had volume ratios <0.8, six Sef-electroporated embryos fell outside this range for NM, and two fell outside this range for NA. D, Dorsal; V, ventral.