Functional Synergy between Cholecystokinin Receptors CCKAR and CCKBR in Mammalian Brain Development

<div><p>Cholecystokinin (CCK), a peptide hormone and one of the most abundant neuropeptides in vertebrate brain, mediates its actions via two G-protein coupled receptors, CCKAR and CCKBR, respectively active in peripheral organs and the central nervous system. Here, we demonstrate that the CCK receptors have a dynamic and largely reciprocal expression in embryonic and postnatal brain. Using compound homozygous mutant mice lacking the activity of both CCK receptors, we uncover their additive, functionally synergistic effects in brain development and demonstrate that CCK receptor loss leads to abnormalities of cortical development, including defects in the formation of the midline and corpus callosum, and cortical interneuron migration. Using comparative transcriptome analysis of embryonic neocortex, we define the molecular mechanisms underlying these defects. Thus we demonstrate a developmental, hitherto unappreciated, role of the two CCK receptors in mammalian neocortical development.</p></div

Pathway analysis (Ingenuity.com) of RNA-Seq data.

<p>Pathway analysis (Ingenuity.com) of RNA-Seq data.</p

Defects in cortical development in <i>Cckar/br</i> mutant mice.

<p>(A and B) Nissl staining of coronal sections of control and <i>Cckar/br</i> mutant brains at postnatal day 14 (P14) reveals thickening of the cingulate cortex, agenesis of the corpus callosum, and lateral displacement of the hippocampus. (C and D) In situ hybridization with <i>claudin 11</i>, which is expressed in myelinated oligodentrocytes thus highlighting white matter tracts, shows that the commissural axons of the corpus callosum do not cross to the contralateral hemisphere in <i>Cckar/br</i> mutants (D) compared to control (C). Of a total of 16 brains from <i>Cckar/br</i> mutants that were fully sectioned and analyzed for callosal development, 13 had complete and 3 had partial agenesis of the corpus callosum. Scale bar, 100 μm. (E and F) Labeling of callosal projections with DiI shows that colossal axons fail to cross the midline in <i>Cckar/br</i> double mutants (F). Green lines (in E,F) and asterisk (in F) indicate the midline and Probst bundles, respectively. Scale bar, 200 μm.</p

Validation of comparative transcriptional profiling findings.

<p>(A-D) <i>Cxcl12</i>, encoding a chemokine that is a known attractant for tangentially migrating interneurons, is downregulated (fold change: -2.104, p = 6.14E-08) in <i>Cckar/br</i> mutants. In situ hybridization demonstrates a reduction in <i>Cxcl12</i> transcripts in the SVZ (arrows) of mutant embryos compared to controls at E14.5 (A,B) and E17.5 (C,D). (E and F) <i>Bmp7</i>, encoding a secreted protein, is also downregulated (fold change: -2.092, p = 2.13E-03) in <i>Cckar/br</i> mutants. Arrows indicate lower expression levels of <i>Bmp7</i> in the midline of the mutants. Scale bar, 200 μm. (G-L) <i>Nrp2</i>, encoding the neuropilin 2 receptor, is upregulated (fold change: 1.510, p = 3.29E-04) in the <i>Cckar/br</i> mutants (H,J,L) compared to controls (G,I,K). The sections shown are at three different rostro-caudal levels (G and H, I and J, K and L). Arrows point to areas of differential expression levels of <i>Nrp2</i>, expanded in the mutants. Scale bar, 200 μm.</p

Defects in tangentially migrating interneurons in <i>Cckar/br</i> mutants.

<p>(A,B,E,F) Interneuron progenitors are generated normally in <i>Cckar/br</i> mutant embryos compared to controls, as demonstrated by in situ hybridization with <i>Gad1</i> (A,B) and <i>Lhx6</i> (E,F). However their tangential migration appears delayed in the mutants. Arrows in (A,B) indicate the onset of tangential routes employed by migrating interneurons. Note a delay/reduction in tangential migration in <i>Cckar/br</i> mutants. Red marks in (E,F) indicate the extent of tangential migration (deep route) in the neocortex. Scale bar (B,D), 200 μm. (C,D,G,H) In situ hybridization with <i>Gad1</i> and <i>Lhx6</i> at birth (P0, C,D) or late embryogenesis (E17.5, G,H) demonstrates that despite the delay in migration, a majority of interneurons settle into the neocortex. Scale bar (F,H), 200 μm.</p

Select transcripts identified by comparative transcriptional profiling.

<p>Select transcripts identified by comparative transcriptional profiling.</p

Expression of CCK and its receptors in mouse and human brain.

<p>(A, B) <i>Cckar</i> is highly expressed in the developing mouse brain at E13.5, contrary to <i>Cckbr</i> mRNA, which is barely detectable at this stage. Scale bar, 200 μm. (C) Select embryonic cells express <i>Cck</i> at E15.5. Scale bar, 500 μm (D and E) Expression of <i>CCKBR</i> (D) and <i>CCK</i> (E) in human fetal neocortex at 20 gestational weeks. Both transcripts are detected in the developing cortical plate (cp); <i>CCK</i> is also expressed in the marginal zone (mz). Scale bar, 0.5 mm. (F-H) By the second postnatal week, <i>Cckar</i> becomes restricted to the hippocampus and select extracortical sites, whereas <i>Cckbr</i> is highly expressed in the neocortex; <i>Cck</i> is also robustly expressed in the neocortex, hippocampus and thalamic nuclei. Scale bar, 100 μm. Note the opposite trends in expression levels of the two receptors at embryonic and postnatal stages. (I and J) Expression of <i>CCKBR</i> (I) and <i>CCK</i> (J) in human adult cortex (temporal lobe). Both transcripts are detected throughout all cortical layers with <i>CCK</i> being more robustly expressed. Scale bar, 0.5 mm.</p

Neuronal migration defects in <i>Cckar/br</i> mutants.

<p>(A-F) In situ hybridization with markers of cortical interneurons at P7: <i>Gad1</i> (A,B) (a general interneuron marker); <i>Lhx6</i> (C,D) (a marker of MGE-derived interneurons); and <i>reelin</i> (E,F) (a marker of CGE- and pre-optic area derived interneurons) indicates a significant overall reduction of interneurons in <i>Cckar/br</i> mutants, irrespective of their origin. Scale bar, 200 μm. (G) Quantification of <i>Lhx6-</i>expressing interneurons. <i>Cckar/br</i> mutants have a 26% reduction in MGE-derived cortical interneurons at P7 (p = 0.0160, n = 3). (H) Distribution of <i>Lhx6-</i>expressing interneurons. Despite the reduction in their number, cortical interneurons are distributed in a similar fashion in control and <i>Cckar/br</i> mutant neocortex, suggesting that interneurons that successfully completed tangential migration are able to respond to local cues in the neocortex and settle into their final position. The fraction of <i>Lhx6-</i>positive interneurons per bin is similar in control and <i>Cckar/br</i> mutant neocortices (p is at least > 0.15 for all comparisons, n = 3). (I) Quantification of <i>reelin</i>-expressing interneurons. <i>Cckar/br</i> mutants have a 15% reduction in CGE- and POA-derived cortical interneurons at P7 (p = 0.0226, n = 4). Error bars represent s.e.m.</p

Abnormal differentiation of the midline in <i>Cckar/br</i> mutant embryos.

<p>(A-F) In situ hybridization with <i>Gfap</i>, a marker of midline glia at perinatal stages, reveals that two midline cell populations, the induseum griseum (IG) and midline zipper glia (mzg), are reduced in <i>Cckar/br</i> double mutants (D-F) compared even with <i>Cckar^+/-;Cckbr^+/-</i> double heterozygous embryos (A-C) at E17.5. Expression of <i>Gfap</i> at the glial wedge (gw) does not appear to be affected to the same extent, suggesting defective differentiation of the midline. Scale bar, 200 μm. (G and H) In situ hybridization with <i>Tag1</i>, a marker of commissural neurons, which also marks the subcallosal sling (scs), demonstrates that a continuous sling is present in control <i>Cckbr^+/-</i> pups (G) at P0, but it fails to transverse the gap between the two hemispheres in <i>Cckar/br</i> mutants (H). Scale bar, 200 μm. (I-N) In situ hybridization demonstrates a modest increase in <i>Cckbr</i> transcripts in the ventral hypothalamus of <i>Cckar</i> single mutants (J,L,N) compared with wild type (I,K,M) at P14. The sections shown are at three different rostro-caudal levels. Scale bar, 0.5 mm.</p