Islet1 sequence analysis and expression in dearisch mice. (a, b) In the wild-type original background mouse, capillary sequencing confirmed a T/T residue (a), while in affected animals C/T was found (b). No homozygote mutants were identified, suggesting homozygote lethality. (c) The thymine base indicated in red was conserved among the species shown and also in giant panda, guinea pig, cow, sloth, armadillo, hedgehog, horse, gorilla, African elephant, mouse lemur, opossum, rabbit, chimp, hyrax, brown bat, common shrew, wild boar, puffer fish, bush baby, dolphin and alpaca (sequences obtained from Ensembl ). (d) Using ConSurf  the tyrosine amino acid residue (indicated by a blue arrow) was found to have a high conservation score of 8, and was predicted to be buried (green letter 'b') rather than exposed (orange letter 'e'). It is not noted as being either structural (blue letter 's') or functional (red letter 'f'); however, it is next to a highly conserved, exposed, functional residue and therefore may be important in positioning this residue. (e) Immunohistochemistry using Isl1 antibody indicates expression (brown) within the mucosal lining of the middle ear cavity (MEC) in wild-type adult mice. (f) Immunohistochemistry showing Isl1 labeling in the cell layer covering the malleus (M) and the outer layer of the tympanic membrane, adjacent to the external auditory canal (EAC) in the wild-type adult. (g) Immunohistochemistry showing more diffuse Isl1 labeling in the cell layer over the malleus at postnatal day 4. The middle ear is still largely filled with mesenchyme (MES) at this early stage. Scale bar: 20 μm (e, f); 40 μm (g).