Fig. 5. Elimination of Brn3a binding to its own enhancer abolishes autoregulation. Embryos expressing a Brn3a-mut/lacZ transgene and varying Brn3a gene dosage were analyzed for ß-gal expression. To ensure that the loss of autoregulation by the mutant enhancer was not an artifact of transgene insertion, two independent Brn3a-mut/lacZ lines were analyzed. (A,B) ß-gal staining is similar in wild-type and knockout embryos in the first of two lines analyzed. ß-gal expression in Brn3a^+/+ embryos (A) is qualitatively indistinguishable from embryos expressing the wild-type Brn3a/lacZ transgene. Abnormal features of the Brn3a^-/- embryos are indicated in B, including defasciculation of axon bundles and aberrant axons (small arrows), an ectopic mass of cell bodies and fibers adjacent to the caudal hindbrain (arrowheads), and abnormal trigeminal innervation of the CNS [the normal extent of trigeminal innervation of the principal (pr5) and spinal (sp5) trigeminal nuclei is outlined]. 5g, trigeminal ganglion; 9g, 9/10 cranial ganglion complex; drg, dorsal root ganglion. (C) A second transgenic line in which ß-gal expression is independent of Brn3a gene dosage for all three Brn3a genotypes. (D) RT-PCR assays of ß-gal mRNA in E13.5 embryonic head demonstrate that transgene expression is only slightly increased in Brn3a knockout embryos.