Bone morphogenetic protein signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior

Abstract

The cortical hem is an embryonic signaling center that generates bone morphogenetic proteins (BMPs) and acts as an organizer for the hippocampus. The role of BMP signaling in hippocampal neurogenesis, however, has not been established. We therefore generated mice that were deficient in Bmpr1b constitutively, and deficient in Bmpr1a conditionally in the dorsal telencephalon. In double mutant male and female mice, the dentate gyrus (DG) was dramatically smaller than in control mice, reflecting decreased production of granule neurons at the peak period of DG neurogenesis. Additionally, the pool of cells that generates new DG neurons throughout life was reduced, commensurate with the smaller size of the DG. Effects of diminished BMP signaling on the cortical hem were at least partly responsible for these defects in DG development. Reduction of the DG and its major extrinsic output to CA3 raised the possibility that the DG was functionally compromised. We therefore looked for behavioral deficits in double mutants and found that the mice were less responsive to fear- or anxiety-provoking stimuli, whether the association of the stimulus with fear or anxiety was learned or innate. Given that no anatomical defects appeared in the double mutant telencephalon outside the DG, our observations support a growing literature that implicates the hippocampus in circuitry mediating fear and anxiety. Our results additionally indicate a requirement for BMP signaling in generating the dorsalmost neuronal lineage of the telencephalon, DG granule neurons, and in the development of the stem cell niche that makes neurons in the adult hippocampus.

Figure 1.

Generating double mutant mice deficient in Bmpr1a and Bmpr1b. A–C, Coronal sections through the telencephalon of control mice at E11.5, processed with in situ hybridization to show expression of Bmpr1a (A), Bmpr1b (B), and a third type I BMP receptor gene, Alk2 (C). Bmpr1a is expressed throughout the telencephalic neuroepithelium including the cortical hem; Bmpr1b is expressed more selectively in the dorsal telencephalon with a hot spot of expression at the hem; Alk2 is expressed broadly in the dorsal and ventral telencephalon, but not in the cortical hem (C, C′). D, Breeding strategy used to generate mice lacking Bmpr1b constitutively and Bmpr1a conditionally, the Bmpr1a^fx/−;Bmpr1b^−/−;Emx1^+/IREScre double mutant genotype. E, PCR analysis of DNA extracted from the tail and telencephalon of E12.5 Bmpr1a^fx/−;Emx1^+/IREScre and Bmpr1a^fx/− mice. Primers fx1 and fx4 (Mishina et al., 2002) amplified a 180 bp fragment from Bmpr1a^fx/−;Emx1^+/IREScre telencephalon (lane 4), indicative of Cre-mediated recombination of Bmpr1a^fx. The 180 bp “recombined” band was not amplified from tail tissue (lane 2) or Bmpr1a^fx/− telencephalon (lane 6). Primers fx3 and fx5 amplified a 190 bp fragment from the Bmpr1a constitutive null allele in all three tissue samples (lanes 1, 3, 5). F, G, Coronal sections through the hem region at E12.5 in a control mouse (c) and a double mutant (dm), immunostained for pSmad1/5/8, transcription factors activated downstream of BMP signaling. The red arrows indicate pSmad1/5/8-IR cells in the control hem (F) but virtually no pSmad1/5/8-IR cells in the double mutant hem (G). Outside the hem, in the hippocampal primordium, pSmad1/5/8-IR cells are dense along the ventricular surface (F, G). H, I, A young adult control mouse (H) and littermate double mutant (I). The double mutant is slightly smaller than the control; the red arrows indicate truncated digits and partial loss of facial hair (see Results). Abbreviations: hem, Cortical hem; hp, hippocampus; lge, lateral ganglionic eminence; mge, medial ganglionic eminence; ncx, neocortex; th, thalamus. Scale bar: (in A) A–C, 200 μm; C′, 100 μm; F, G, 50 μm.

Figure 2.

Loss of function of two BMP receptor genes leads to a reduced DG. A–G, Coronal sections through the hippocampus of 8-week-old (A–C) and 4-week-old (D–G) mice. A–C, Sections stained for NeuN immunoreactivity from mice lacking Bmpr1b and heterozygous for Bmpr1a (A), lacking Bmpr1a conditionally and heterozygous for Bmpr1b (B), or lacking both Bmpr1a and Bmpr1b (C). Only the double mutant shows a reduced gcl (C). D–G, Calbindin immunoreactivity reveals that the entire DG including the gcl, molecular layer (mol), hilus (h), and the SGZ is smaller in double mutants compared with controls. H, The cross-sectional area of the gcl in double mutants is ∼60% of that in control mice; this proportion is not significantly different at anterior, mid, or posterior levels of the DG. Data are represented as means ± SEM. Scale bar: (in G) A–G, 375 μm.

Figure 3.

A reduced DG is associated with a drop in neonatal but not adult neurogenesis. A, B, Proliferating cells labeled with immunohistochemistry for BrdU (A, arrows) or PH3 in 8-week-old brains. BrdU-IR cells appear equally dense in the SGZ and hilus in control and double mutant brains (A). The rectangles in A indicate field size used for counting BrdU-IR and PH3-IR cells. B, Density of PH3-IR cells in the SGZ of control and double mutant brains (n = 3, each group) is not significantly different. C, Coronal sections through the DG hilus at P5 processed with double immunofluorescence for BrdU and the neuronal marker NeuN. Double immunofluorescence (merge) labels twice as many newborn neurons in the DG hilus of a control mouse than a double mutant (the white arrows indicate double-positive cells). D, Density of PH3-IR cells in the hilus of neonatal control and double mutant brains (n = 3, each group). The density of mitotic cells was significantly less in double mutants (p = 0.003, one-tailed t test). Data are represented as means ± SEM. **p < 0.01. Scale bar: (in C) A, 100 μm; C, 50 μm.

Figure 4.

The projection from the DG to CA3 is abnormal in double mutants. A–H, Coronal sections through 8-week-old brains, processed for calbindin (A–D) class III β-tubulin (E, F) or NeuN immunoreactivity (G, H). A–D, In double mutant brains, the infrapyramidal mossy fiber bundle (imf) is truncated (A, B, arrowheads), and the intrapyramidal and suprapyramidal mossy fibers (grouped as smf) are reduced (A–D, white bars in C, D). E–H, The stratum lucidum of the hippocampus (E, F, broken outline) is filled with mossy fiber-recipient apical dendrites (ad) of CA3 neurons. CA3 apical dendrites are shorter in double mutants compared with controls (E, F, red lines); the arrows indicate the proximal parts of the apical dendrites of two neurons in G and H. Abbreviation: pcl, Pyramidal cell layer. Scale bar: (in C) A, B, 300 μm; C–F, 75 μm; G, H, 30 μm.

Figure 5.

DG development is disrupted from its inception. A–H, Coronal sections through the telencephalon processed for in situ hybridization. A–D, E14.5. E–H, P0. A–D, The pDG and hem express lower levels of Id3 and Msx2, genes upregulated by BMP signaling, in a double mutant (B, D) compared with a control (A, C). The pDG domain is indicated with double arrows (A, B). E–H, The secondary DG progenitor cell pool migrates toward the forming DG, together with differentiating DG neurons. Both cell types express Cxcr4, and differentiating neurons express Prox1. Expression of Cxcr4 in the double mutant brain indicates a narrower band of migratory cells (E, F, arrowheads), and expression of Prox1 reveals a greatly reduced population of migrating, differentiating DG neurons (G, H, arrowheads). Scale bar: (in A) A, B, 150 μm; C–H, 100 μm.

Figure 6.

Defects of Wnt signaling in the cortical hem. A–G, Coronal sections through the E12.5 telencephalon processed for in situ hybridization. A, Expression of Fz10 in wild type (wt) and heterozygous and homozygous Wnt3a mutant brains. Fz10 expression fills the wild-type hem and appears in patches in the CPe; expression is weaker in the heterozygous Wnt3a mutant hem and absent from the homozygous mutant hem. B–E, The hem shows a smaller domain of Wnt3a expression in a double mutant compared with a control brain (B, C) and greatly reduced expression of Fz10 (D, E). F, G, A region (between arrows) including the hem and pDG shows immunoreactivity for a Wnt antagonist, DKK1. DKK1 immunoreactivity is more intense in this region in the double mutant (G) than in the control (F). Scale bar: (in A) A–E, 200 μm; F, G, 100 μm.

Figure 7.

Double mutant mice show diminished fear conditioning and lower anxiety. A, Freezing behavior in the shock chamber preceding the 2 s footshock (arrowhead) and for 30 s afterward. Freezing behavior did not differ in the training trial between double mutants and control mice. B, C, Double mutant mice showed significantly reduced responses in retention tests of contextual and cued fear learning. Tested 24 h after the single conditioning trial, double-mutant mice froze significantly less than control mice in response to the conditioned stimulus (contextual, p = 0.04; cued, p = 0.0008). D, E, In an elevated plus maze, double mutant mice spent significantly more time in the open arms than did control mice (p = 0.0007) and made more open arm entries as a percentage of total entries (p = 0.0008). There was no significant difference in the number of total entries between the two groups (p = 0.7). *p < 0.05; ***p < 0.001. Data are represented as means ± SEM.