Supplemental Figure 1. Egr2Cre and Hoxb1Cre are expressed in and delete Atoh1flox from different regions of the developing neural tube. (A, A’) E9.5 Egr2Cre; ROSAR26R embryo wholemount stained with Xgal. Cre expression drives β-galactosidase expression only in rhombomeres 3 and 5. White and black dotted lines here and in panels E and E’ denote the anterior and posterior boundaries of rhombomere 4, respectively. (B, C) Coronal sections from a P30 Egr2Cre; ROSAR26R animal showing β-galactosidase expression in the CN. Large cells and granule cells are labeled in the AVCN and DCN, but to a much lesser extent in the PVCN. (D) Xgal staining in coronal sections of the AAN of the same animal. High levels of expression are seen throughout the AAN, demonstrating that these nuclei arise predominantly from rhombomeres 3 and 5. (E, E’) E9.5 Hoxb1Cre; ROSAR26R embryo wholemount stained with Xgal. Cre expression drives β-galactosidase expression posterior to the anterior boundary of rhombomere 4. (F, G) Coronal sections from a P30 Hoxb1Cre; ROSAR26R animal showing heavy expression of β-galactosidase in the PVCN and DCN with much lower levels of expression in the AVCN. (H) Xgal staining in coronal sections of the AAN of the same Hoxb1Cre; ROSAR26R animal. High levels of expression are seen throughout the AAN with the exception of the MNTB, where only some of the neurons are labeled. (I-N) Atoh1 in situ hybridization in coronal hindbrain sections from E15.5 wildtype (I, L), Egr2Cre; Atoh1CKO (J, M) and Hoxb1; Atoh1CKO (K, N) embryos. Red arrows show the absence of Atoh1 mRNA in the neuroepithelium that contributes neurons to the developing AES, AVCN, cRL, and DCN, while expression of the gene is maintained in other regions where Cre is not expressed. AES – anterior extramural migratory stream, AVCN – anteroventral cochlear nucleus, CB - cerebellum, cRL – caudal rhombic lip, DCN – dorsal cochlear nucleus, EGL – external granular layer, LSO – lateral superior olive, MNTB –medial nucleus of the trapezoid body, MSO – medial superior olive, nVII – 7th nerve, PVCN – posterventral cochlear nucleus, r3, r4, r5 – rhombomeres 3-5, SPN – superior periolivary nucleus, Sp V – spinal trigeminal nucleus. Scale bars: (A, E) 1 mm, (A’, E’) 200 μm, (B-D, F-H) 200 μm, (I-N) 500 μm.

Supplemental Figure 2. The AVCN is lost in Egr2; Atoh1CKO animals. Complete series of Cresyl violet stained coronal sections spaced approximately 60 μm through the brainstems of 6 week old wildtype and Egr2; Atoh1CKO animals. The sections are matched for anteroposterior level with the rostral-most sections in the upper left and the caudal-most sections in the lower right of the figure. Brainstem landmarks are labeled to facilitate comparison between the series. ** represents the absence of cells where the AVCN should be located in the Egr2; Atoh1CKO animal. AAN – accessory auditory nuclei, AVCN – anteroventral cochlear nucleus, DCN – dorsal cochlear nucleus, Fl – flocculus, Lat – cerebellar nucleus lateralis, MeVe – medial vestibular nucleus, nVII – 7th nerve, nVIII – 8th nerve, PrV – principle trigeminal nucleus, PVCN – posteroventral cochlear nucleus, VII – facial motor nucleus. Scale bar: 500 μm.

Supplemental Figure 3. The PVCN and most of the DCN are lost in Hoxb1; Atoh1CKO animals. Complete series of Cresyl violet stained coronal sections spaced approximately 60 μm through the brainstems of 6 week old wildtype and Hoxb1; Atoh1CKO animals. The sections are matched for anteroposterior level with the rostral- most sections in the upper left and the caudal-most sections in the lower right of the figure. Brainstem landmarks are labeled to facilitate comparison between the series. AAN – accessory auditory nuclei, AVCN – anteroventral cochlear nucleus, DCN – dorsal cochlear nucleus, Fl – flocculus, Lat – cerebellar nucleus lateralis, MeVe – medial vestibular nucleus, nVII – 7th nerve, nVIII – 8th nerve, PrV – principle trigeminal nucleus, PVCN – posteroventral cochlear nucleus, VII – facial motor nucleus. Scale bar: 500 μm.

Supplemental Figure 4. Neurons are lost from the AAN of Egr2; Atoh1CKO and Hoxb1; Atoh1CKO animals. Complete series of Cresyl violet stained coronal sections spaced approximately 60 μm through the brainstem of 6 week old wildtype, Egr2; Atoh1CKO and Hoxb1; Atoh1CKO animals. Brainstem landmarks are labeled to facilitate comparison between the series. All subnuclei of the AAN are affected in Egr2; Atoh1CKO animals, while loss is seen only in the LSO and MSO of Hoxb1; Atoh1CKO animals. LSO – lateral superior olive, MNTB – medial nucleus of the trapezoid body, MSO – medial superior olive, nVII – 7th nerve, PN – nucleus basis pontis, PrV – principle trigeminal nucleus, RPN – rostral periolivary nucleus, RTGN – reticulotegmental nucleus, SPN – superior periolivary nucleus, VII – facial motor nucleus, VNTB – ventral nucleus of the trapezoid body. Scale bar: 200 μm.