ImmunohistochemistryVibratome and cryosections were blocked in 5% BSA/1% Tween (SigmaAldrich) in PBS (pH 7.4) for 1 hour at room temperature. Mouse monoclonal anti-RC2 (IgM) antibody (Developmental Hybridoma Bank) , which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.

Necdin, a member of the MAGE (melanoma antigen) protein family, is expressed predominantly in terminally differentiated neurons. The necdin gene NDN is maternally imprinted and expressed only from the paternal allele, the deficiency of which is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Necdin binds to its homologous MAGE protein MAGE-D1 (also known as NRAGE or Dlxin-1), which interacts with Msx (msh homeobox) and Dlx (distal-less homeobox) family homeodomain transcription factors. Members of the Dlx homeobox gene family are involved in the differentiation and specification of forebrain GABAergic neurons. Here we demonstrate that necdin associates with Dlx homeodomain proteins via MAGE-D1 to promote the differentiation of GABAergic neurons in mouse embryonic forebrain. Immunohistochemical analysis revealed that necdin was coexpressed with Dlx2, Dlx5, or MAGE-D1 in a subpopulation of embryonic forebrain cells. Necdin bound to Dlx2 and Dlx5 via MAGE-D1 and enhanced Dlx2-dependent activation of the Wnt1 (wingless-type MMTV integration site family) promoter. Necdin significantly increased the populations of cells expressing the GABAergic neuron markers calbindin D-28k and glutamic acid decarboxylase when overexpressed by electroporation in cultured forebrain slices. In this assay, Dlx5N, a truncated Dlx5 mutant that competes with Dlx2 to bind MAGE-D1, diminished the effect of necdin on GABAergic neuron differentiation. Furthermore, mutant mice lacking the paternal necdin allele showed a significant reduction in the differentiation of forebrain GABAergic neurons in vivo and in vitro. These results suggest that paternally expressed necdin facilitates the differentiation and specification of GABAergic neurons in cooperation with Dlx homeodomain proteins.

Summary At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here we identify a tripartite molecular system for contralateral RGC projections: Semaphorin 6D and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth-promotion. Nr-CAM functions as a novel receptor for Sema6D. Sema6D, Plexin-A1 and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a novel mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.

Summary The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live-imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. As Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2−/− mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation.