Since these IRs are not significantly altered by DT treatment in the dorsal or ventral hilus in controls (Figure S2B), and there is no difference among control genotypes in number of hilar GluA2/3-, CR-, and neuropeptide Y (NPY)-positive cells (Figure S2C),

we combined data from our three control genotypes (Cre, fDTR, and B6 wild-type) to form our combined control (control) group. In mutants 1 week after DT treatment, the number of GluA2/3-positive cells in the hilus of the dorsal hippocampus decreases to 26.1%, and by 4 weeks after Selleck LDN193189 treatment to 9.5% compared to numbers in DT-treated controls (Figures 3B to 3D). Similarly, the number of GluA2/3-positive cells in the ventral hilar region in mutants decreases selleck compound to 27.9% of that in controls by week one and to

10.5% by week four following DT treatment. The number of CR-positive hilar cells in mutants decreases by week one to 79.9% and by week four to 6.7% compared to levels found in controls. By contrast, 4 weeks after DT treatment mutants show no obvious effect on the interneuron marker NPY-IR in the dorsal or ventral hilus (Figures 3C and 3D). Different rates of reduction in GluA2/3- and CR-positive hilar cells following DT treatment may arise from variability in protein degradation. While GluA2/3- and CR-positive mossy cells mostly overlap (Figure S2A; see Fujise et al., 1998), 1 week after DT treatment the number of GluA2/3- and CR-positive ventral

hilar cells originating from the same mutant out brain tissues varies widely (Figure 3B). In contrast, DT treatment does not obviously affect the interneuron marker NPY-IR in the dorsal or ventral hilus in mutants (Figures 3C and 3D). Since hilar neurodegeneration is already prominent one week after DT treatment (Figures 2 and 3A), loss of GluA2/3 mossy-cell-marker labeling is likely to be a signature of mossy cell neurodegeneration. If so, our results show that in mutants, ∼75% of mossy cells are selectively degenerated 7 days after DT treatment and ∼90% by 4 weeks post-DT. To assess the acute effects of functional mossy cell loss, we performed experiments at 4–11 days (acute phase), and to assess the chronic effects, at 4–6 weeks (chronic phase) after DT treatment. Cre-recombination also occurs in CA3c pyramidal neurons (Figures 1A and S1A), whose axons may project, either directly or via mossy cells, to dentate granule cells (Scharfman, 2007; Wittner et al., 2007).

For these

new gene expression profiling experiments, we chose P0 as the time point, both for practical reasons MLN2238 chemical structure and with the hope of discovering new target genes. Interestingly, we found that just as Prdm8 mRNA is upregulated in Bhlhb5 mutant mice, so Bhlhb5 mRNA is upregulated in Prdm8 mutant mice ( Figures 4A and 4B). But what about other Bhlhb5 target genes—are they likewise upregulated in Prdm8 mutant mice? We found that loss of either Bhlhb5 or Prdm8 resulted in changes in gene expression in a small number of genes and, remarkably, all of the genes that were significantly upregulated in one mutant were also upregulated in the other. These genes include Antxr2, Connexin36, NMDA3A, and Paqr3 ( Figures 4C–4F) as well as Fgf5 and Netrin1 (data not shown). We therefore conclude that Bhlhb5 and Prdm8 inhibit the expression of a common set of genes, consistent with the possibility that they function together as part of the same repressor complex. We next investigated more Ponatinib directly the possibility that Bhlhb5 and Prdm8 form a

repressor complex by characterizing Bhlhb5 occupancy throughout the genome and testing whether Prdm8 is bound to the same genomic loci as Bhlhb5. The genomic binding sites of Bhlhb5 in the dorsal telencephalon were mapped by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). As a negative control, we also performed ChIP-seq using tissue from Bhlhb5−/− heptaminol mice, thereby confirming the specificity of the Bhlhb5 antibody. In these ChIP-seq experiments, we identified ∼2,300 specific Bhlhb5 binding sites, representing approximately one binding site per million bases (see ftp://ross-et-al-2012.hms.harvard.edu to visualize genomic data online). In addition to describing Bhlhb5 binding sites in the brain across the genome, the identification of these sequences allowed us to uncover an eight nucleotide consensus binding motif for Bhlhb5: CATATGNTNT ( Figure 5A). Thus, Bhlhb5 binds to a sequence element consisting of a canonical E-box (underlined),

a motif common to many members of the basic helix-loop-helix family, together with several other key nucleotides that likely confer additional sequence specificity. Having identified genomic Bhlhb5 binding sites in the brain, we were in a position to ask whether Prdm8 binds to the same DNA sequence elements. To address this question, we chose several genes from the ChIP-seq data to test, including two genes that showed Bhlhb5 binding in the proximal promoter, Bhlhb5 itself and Repressor Protein 58 (RP58) ( Figures 5B and 5C). In addition, we selected one of the putative Bhlhb5 target genes identified by expression profiling, Cdh11, which showed Bhlhb5 binding within its first intron ( Figure 5D).