A) Absence of the corticospinal tract in the spinal cord of mice lacking either Bhlhb5 or Prdm8. In wild type mice (WT), PKCγ immunostaining labels the axons from corticospinal motor neurons as they descend in the dorsal funiculus of the spinal cord (white arrow). In both Bhlhb5−/− and Prdm8−/− mice, this axon tract is absent at all levels of the spinal cord. Similar results are observed when this fiber tract is labeled genetically (data not shown). Note that PKCγ also labels a subset of lamina II neurons in the dorsal horn, which are unaffected by the loss of Bhlhb5 or Prdm8. Images are of representative cervical spinal cords from adult mice (8 wks). B) Agenesis of the corpus callosum and hippocampal commissure in mice lacking either Bhlhb5 or Prdm8. In wild type mice (WT), dense bundles of axons connect the two hemispheres of the dorsal telencephalon (black arrow). In Bhlhb5−/− and Prdm8−/− mice, there is severe agenesis of these colossal fiber tracts. This axon targeting phenotype was observed in 20/20 Bhlhb5−/− mice, 5/7 Prdm8−/− mice, and none of the corresponding wild type littermates. Images are of representative coronal brain sections from adult mice stained with luxol fast blue and cresyl violet. C) Mice lacking either Bhlhb5−/− or Prdm8−/− show elevated scratching behavior that results in the development of skin lesions. Photos illustrating characteristic lesions are shown. All Bhlhb5−/− mice develop skin lesions at approximately 6 wks of age; ~75% of Prdm8−/− mice eventually develop skin lesions. D) Both Bhlhb5−/− and Prdm8−/− mice show an unusual movement in which they walk on their forepaws. Note that this ‘handstand’ phenotype, which appears to be secondary to abnormal contraction of the hindpaws, is only observed in a small fraction (~1– 5%) of Bhlhb5 or Prdm8 mutants. Also see Figure S2 for a more detailed analysis of axon targeting defects and Figure S3 for analysis of cortical lamination, which is unaffected by the loss of either Bhlhb5 or Prdm8.