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. 2017 Sep 26;11:307.
doi: 10.3389/fncel.2017.00307. eCollection 2017.

Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain

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Free PMC article

Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain

Andrea Forero et al. Front Cell Neurosci. .
Free PMC article

Abstract

Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.

Keywords: T-cadherin; cadherin-13 (CDH13); dorsal raphe; neurodevelopment; prefrontal cortex; psychiatric disorders; radial glia; serotonin.

Figures

Figure 1
Figure 1
Cdh13 in embryonic mouse brain at selected developmental stages. (A) Cdh13 immunoreactivity is prominent in the hindbrain at E13.5, extends to various brain regions including the cortex at E15.5 and displays a layer-associated expression in the cortex at E17.5. (B) Cdh13 is not localized in regions immunoreactive for Nestin (yellow arrow: area positive for Nestin). (C) Cdh13 is found in the hindbrain at E13.5 in two groups of fibers that extend longitudinally and transversally (D) Cdh13 immunoreactivity in the hindbrain is juxtaposed to Otx2 midbrain staining, delimiting the MHB at E13.5 (yellow dotted line: MHB). Orientation: sagittal. Scale bars in (A) 500 μm, in (B) 200 μm, in (C,D) 100 μm. Ctx, cortex; Hb, hindbrain; Mb, midbrain; Th, thalamus; OB, olfactory bulb.
Figure 2
Figure 2
Cdh13 in 5-HT neurons of the dorsal raphe. (A) Cdh13 is highly expressed in the DR at E13.5. Cdh13 and 5-HTT-ir fibers follow the same trajectory from the hindbrain to the forebrain at E15.5 (B) and E17.5 (C). Orientation: sagittal. Scale bars: 100 μm. DR, dorsal raphe; TH, thalamus; HTH, hypothalamus; mlf, medial longitudinal fasciculus.
Figure 3
Figure 3
Cdh13 is not expressed in fibers in the median raphe. Representative images of the hindbrain region stained for Cdh13 and 5-HT. Cdh13 is not detected in the region corresponding to the developing MR (delimited area). Arrow indicates position of DR. Orientation: sagittal. Scale bar: 100 μm.
Figure 4
Figure 4
Subcellular localization of Cdh13 in DR 5-HT neurons. Using SIM, Cdh13 is detected in the soma (A) and neurites (B) of 5-HT positive neurons, and is also localized in 5-Htt positive fibers (C) (yellow arrows indicate points of colocalization). Orientation: sagittal. Scale bars in (A) 100 μm, in (B,C) 10 μm in full images, 2 μm in the magnified boxed regions.
Figure 5
Figure 5
Cdh13 and 5-HT in prefrontal cortex at E17.5. (A) Cdh13 is expressed in cortex, including the PFC (delimited by a white rectangle). (B) In this region, 5-HT specific afferents innervate the intermediate (IZ) and marginal zones (MZ), both of which exhibit high immunoreactivity for Cdh13. Orientation: sagittal. Scale bar: 100 μm. IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; OB, olfactory bulb.
Figure 6
Figure 6
Cdh13 in radial glia cells in the dorsal raphe at E13.5. Cdh13 overlaps with RC2 immunoreactivity, a marker for radial glia cells. Orientation: sagittal. Scale bar: 100 μm.
Figure 7
Figure 7
Cdh13 at points of intersection between 5-HT neurons and radial glial cells. Representative images of serotonergic fibers and radial glial cell (RGC) extensions triple-stained for 5-HT, RC2 and Cdh13. (A,B) Cdh13 (green) is present in both 5-HT neurons (red) and RGCs (blue), and at some points of intersection between these two cell types (yellow arrows). (B) Reconstruction of triple IF of 5-HT, RC2, and Cdh13 using Imaris. The 5-HT neuron (cyan arrow) is intertwined with the RGC fiber (magenta arrow). Cdh13 immunoreactivity is found at the interface between both cell types (Supplemental Videos 1 and 2). Orientation: sagittal. Scale bars in (A) 5 μm in full images, 2 μm in the magnified boxed region. FP, floor plate; VZ, ventricular zone.
Figure 8
Figure 8
Cdh13 deficiency affects the cell density and area of the dorsal raphe, but not the median raphe, at E13.5. (A) Representative images of DR stained with 5-HT are shown. A significantly higher number of serotonergic neurons (P = 0.0014) within a predetermined area (397.5 μm2) and a trend toward reduced DR area (P = 0.051) is observed in Cdh13−/− mice compared to wildtype controls. n = 8 per genotype. (B) Representative images of MR stained with 5-HT are shown. No significant differences in the density of serotonergic neurons in the MR between Cdh13−/− and wildtype embryos is observed. n = 4 per genotype. Orientation: sagittal. Scale bar in (A,B): 100 μm. Data are presented as mean ± s.e.m. #P < 0.1, **P < 0.01.
Figure 9
Figure 9
Cdh13 deficiency affects the cell density of the dorsal raphe at E17.5 and adulthood. Representative images of DR stained with 5-HT are shown for E17.5 (A) and adult (B) brains. A significantly higher density of serotonergic neurons is observed in Cdh13−/− mice compared to wildtype controls at E17.5 (A), P = 0.0227 and adult (B), P = 0.0191 brains. N = 6 per genotype. Orientation in E17.5 sections: sagittal. Orientation in adult sections: coronal. Scale bar: 100 μm. Data are presented as mean ± s.e.m. *P < 0.05.
Figure 10
Figure 10
Cdh13 deficiency affects the serotonergic innervation of the prefrontal cortex at E17.5, but not in adulthood. (A) Representative images of PFC at E17.5 (stained with 5-HT) are shown. A larger area occupied by 5-HT-positive fibers is observed in Cdh13−/− mice compared to wildtype controls (P = 0.042). N = 7 per genotype. (B,C) No significant differences in the serotonergic innervation (stained with 5-htt) of the IL and CG cortex, measured by two different approaches (see Methods section), is observed in adult brains. Orientation in E17.5 sections: sagittal. Orientation in adult sections: coronal. Scale bar in (A,B) 100 μm. Data are presented as mean ± s.e.m. *P < 0.05.

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