Molecular features distinguish ten neuronal types in the mouse superficial superior colliculus

Abstract

The superior colliculus (SC) is a midbrain center involved in controlling head and eye movements in response to inputs from multiple sensory modalities. Visual inputs arise from both the retina and visual cortex and converge onto the superficial layer of the SC (sSC). Neurons in the sSC send information to deeper layers of the SC and to thalamic nuclei that modulate visually guided behaviors. Presently, our understanding of sSC neurons is impeded by a lack of molecular markers that define specific cell types. To better understand the identity and organization of sSC neurons, we took a systematic approach to investigate gene expression within four molecular families: transcription factors, cell adhesion molecules, neuropeptides, and calcium binding proteins. Our analysis revealed 12 molecules with distinct expression patterns in mouse sSC: cadherin 7, contactin 3, netrin G2, cadherin 6, protocadherin 20, retinoid-related orphan receptor β, brain-specific homeobox/POU domain protein 3b, Ets variant gene 1, substance P, somatostatin, vasoactive intestinal polypeptide, and parvalbumin. Double labeling experiments, by either in situ hybridization or immunostaining, demonstrated that the 12 molecular markers collectively define 10 different sSC neuronal types. The characteristic positions of these cell types divide the sSC into four distinct layers. The 12 markers identified here will serve as valuable tools to examine molecular mechanisms that regulate development of sSC neuronal types. These markers could also be used to examine the connections between specific cell types that form retinocollicular, corticocollicular, or colliculothalamic pathways. J. Comp. Neurol. 524:2300-2321, 2016. © 2016 Wiley Periodicals, Inc.

Keywords: RRID: AB_10000240; RRID: AB_10000344; RRID: AB_1288870; RRID: AB_2079751; RRID: AB_2167523; RRID: AB_2298772; RRID: AB_305869; RRID: AB_518614; RRID: AB_732196; RRID: AB_91338; RRID: nif-0000-00509; RRID: nif-0000-30467; calcium binding proteins; cell adhesion molecules; neuropeptides; retinal ganglion cells; transcription factors.

Figure 1. Visual layer boundaries of mouse superior colliculus (SC)

A. A schematic shows the labeling of the contralateral superficial part of the SC after cholera toxin b subunit (CTB) injection into one eye (magenta). Vibratome sections were subsequently prepared at ~P22 and immunostained. B. A section stained with anti-myelin basic protein (MBP, green) and CTB. The dashed line delineates the pial surface. A boundary between stratum griseum superficiale (SGS) and stratum opticum (SO) is delineated by the boundary between intense labeling of myelinated fibers (B1) and dense labeling of CTB (B2). From the merged image (B3), a higher magnification of the boxed area is shown (B4). The question mark indicates the inability to locate the boundary between SO and stratum griseum intermediale (SGI) based on MBP labeling. C. A section stained with anti-choline acetyltransferase (ChAT, green). The dashed line delineates the pial surface. A boundary between SO and SGI is delineated by the boundary between intense labeling of ChAT-positive fibers (C1) and the deepest retinal fiber labeled by CTB (C2). From the merged image (C3), a higher magnification of the boxed area is shown (C4). The question mark indicates the inability to locate the boundary between SGS and SO based on ChAT labeling. D. Schematic of layer distinctions based on CTB labeling (magenta), MBP staining (green) and ChAT staining (yellow). The dashed lines and question marks indicate the inability to locate certain boundaries. Scale bar: 200 μm (B1-B3 and C1-C3), 100 μm (B4 and C4).

Figure 2. Expression patterns of three transcription factors

A. A schematic sagittal section of the mouse brain. The boxed area indicates the location where experiments were conducted (~570 ± 140 μm from anterior edge) on the superficial part of the SC (sSC). Also shown are the inferior colliculus (IC), cortex (CTX) and suprachiasmatic nucleus (SCN). A← →P, anterior to posterior axis. B–D. In situ hybridization with each probe was conducted at P10-P13 (left) and P18-P20 (right). The relative position of putative stratum griseum superficiale (pSGS) and putative stratum opticum (pSO) are indicated by brackets. B. Expression of Rorβ is confined to a superficial layer of sSC. C. Expression of Brn3b is confined to a deep layer. Within the region of expression, Brn3b formed two distinct bands in the lateral sSC (shown, arrows); these bands became less distinct in the medial sSC (data not shown). D. Expression of ETV1 is confined to two distinct bands (arrows): a sparse upper band and a denser lower band. In all cases, expression patterns were similar at ~P10 and ~P18. Scale bar: 200 μm.

Figure 3. Expression patterns of five cell adhesion molecules

A–E. In situ hybridization with each probe was conducted in the sSC at ~P10 (left) and ~P18 (right). The positions of pSGS and pSO are indicated by brackets. A. Expression of cadherin 7 (Cdh7) was predominantly confined to a superficial layer of sSC. B. Expression of netrin G2 (Ntng2) was found in both superficial and deep layers, with a gap in between. C. Expression of contactin 3 (Cntn3) was most abundant in a superficial layer. D. Expression of cadherin 6 (Cdh6) was detectable in both superficial and deep layers at ~P10. The expression in the deep layer decreased at ~P18. E. Expression of Pcdh20 was found in both superficial and deep layers, with a gap in between at ~P10. The expression in the deep layer decreased at ~P18. Scale bar: 200 μm.

Figure 4. Expression patterns of four neuropeptides/calcium binding proteins

A–C. In situ hybridization with each probe was conducted in the sSC at ~P10 (left) and ~P18 (right). The positions of pSGS and pSO are indicated by brackets. A. Expression of substance P (SP) was predominantly found in a superficial layer of sSC. B. Expression of somatostatin (SST) was found in middle and deep layers. C. Expression of vasoactive intestinal polypeptide (VIP) was barely detectable at ~P10 but was found in a superficial layer at ~P18. D. Expression of parvalbumin (PV), detected by immunostaining, was sparse at ~P10 but became abundant in a superficial layer, well below the pial surface, at ~P18. Scale bar: 200 μm.

Figure 5. Relative distribution of nine markers compared to expression pattern of SST

A–J. Double fluorescent in situ hybridization (dFISH) was conducted in sSC at ~P10 using SST as a common second probe (labeled with fluorescein, green) and counterstained with DAPI (blue). The first probes (labeled with digoxigenin) are shown in red. The dashed line delineates the pial surface. Relative positions of four layers, defined by the collective expression patterns (see part K), are indicated by the brackets to the right of the combined images. A. Two in situ probes to SST (A1 and A2) overlapped well, which validates the use of SST in subsequent panels (merged, A4; DAPI alone, A3). B. Expression of Cdh7 (B1, red) is found above the region of SST expression (B2, merged). C–G. Expression of Ntng2 (C1), Cntn3 (D1), Pcdh20 (E1), Cdh6 (F1) and Rorβ (G1) are also primarily located above the region of SST expression (C2, D2, E2, F2 and G2; merged). H–I. Expression of Brn3b and ETV1 (H1 and I1) are primarily located within and below the region of SST expression (H2 and I2, merged). The upper band of ETV1 expression was not used as a reference for the layer distinctions because of the sparse labeling. J. Expression of SP (J1) is predominantly located above the region of SST expression (J2, merged). K. Schematic showing layer-enriched expression of each molecule. Brackets show layers 1–4, as distinguished by molecular markers; a dashed line shows the boundary between pSGS and pSO. Scale bar: 200 μm.

Figure 6. Direct comparison of expression patterns for Brn3b and ETV1

Sections of sSC at ~P10 were stained with antibodies to both Brn3b (green, A) and ETV1 (red, B) and counterstained with DAPI (blue). The merged image is shown in C. The boxed area in C is shown at higher magnification in D1 (Brn3b), D2 (ETV1) and D3 (merged). Both bands of Brn3b+ and ETV1+ cells were visible. Individual cells were labeled by either one (red or green) or both antibodies (yellow). Layers 3 and 4 together comprise pSO (Figure 5K). Scale bar: (A–C) 200 μm, (D) 50 μm.

Figure 7. Relationship between sSC layers defined by either sSC cell markers or retinal ganglion cell axons

Vibratome sections at ~P18 were immunostained with specified antibodies (green) after injection of CTB (magenta) into the contralateral eye. A. A sagittal section of sSC labeled with CTB. The pial surface is delineated by a dashed white line, the lower border of stratum griseum superficiale (SGS) by a dashed yellow line and the lower border of stratum opticum (SO) by a dashed green line. B1. A section labeled with anti-SP (green). The retinorecipient layers of sSC are indicated by dashed lines based on CTB labeling, as in A. The higher magnification of the boxed area is shown in B2 (SP) and B3 (merged; SP and CTB labeling). C–F. Same format as B. but with anti-SST, anti-PV, anti-Brn3b and anti-ETV1. G. The schematic shows relative positions of sSC layers defined by either sSC cell markers (layers 1–4) or RGC axons (SGS, SO). Scale bar: (A, B1, C1, D1, E1 and F1) 200 μm, (B2-B3, C2-C3, D2-D3, E2-E3 and F2-F3) 100 μm.

Figure 8. Rorβ marks a subpopulation of sSC neurons

A. Expression of Rorβ was detected by anti-GFP in the Rorβ^1g/+ mouse (see Methods). All images in this figure were acquired in layer 1. Sections of sSC at ~P10 were stained with antibodies to NeuN (red, A1) and GFP (green, A2) and counterstained with DAPI (A3). The merged image (A4) shows that Rorβ+ cells are neurons (i.e., NeuN+; arrows). B–H. Double labeling was conducted at ~P10 in the Rorβ^1g/+ mouse by ISH (first probes, digoxigenin, red) and immunostaining (second probe, anti-GFP, green) and counterstained with DAPI (blue). Arrows indicate overlapping expression and arrowheads indicate expression by the first probe only. Arrows are missing in G and H because overlapping expression between the two genes is rare (<5%). B. An in situ probe to Rorβ (B1, red) and anti-GFP signal (B2, green) highly overlapped (B4, merged; B3, DAPI alone). C–E. The majority of cells expressing Cntn3 (C1), Ntng2 (D1) and Cdh7 (E1) were Rorβ+ (merged; C2, D2, E2). F–H. Very few cells expressing Cdh6 (F1), Pcdh20 (G1) and SP (H1) were Rorβ+ (merged; F2, G2, H2). Scale bar: 20 μm.

Figure 9. Ntng2 marks a subpopulation of Rorβ+ neurons in sSC

A–G. The dFISH was conducted in sSC at ~P10 using Ntng2 as a second probe (labeled with fluorescein, green) and counterstained with DAPI (blue). The first probes (labeled with digoxigenin) are shown in red. All images in this figure were acquired in layer 1. A. Two in situ probes to Ntng2 (A1 and A2) highly overlapped (merged, A4; DAPI alone, A3). Here and in subsequent panels, arrows indicate examples of cells with overlapping expression, and arrowheads indicate expression by the first probe only. Arrows are missing in F and G because overlapping expression between the two genes is rare (<8%). B–D. Approximately half of Rorβ+ (B1), Cntn3+ (C1) and Cdh7+ (D1) cells were Ntng2+ (merged; B2, C2, D2). E–G. Very few cells expressing Cdh6 (E1), Pcdh20 (F1) and SP (G1) were Ntng2+ (merged; E2, F2, G2). Scale bar: 20 μm.

Figure 10. Adult expression of selected molecules used for cell type classification

A–F. In situ hybridization was conducted in the sSC at P30 for six probes: Rorβ (A), Ntng2 (B), Cdh7 (C), SST (D), Brn3b (E) and ETV1 (F). Two distinct bands of Brn3b or ETV1 expression are indicated by arrows. The positions of layers 1–4 are indicated by brackets. G–H. The number of Rorβ+ (G) and SST+ cells (H) remains consistent between P10 and P30. Rorβ+ cells were identified by GFP signals in the Rorβ^1g/+ mouse and counted within layer 1. SST+ cells were analyzed by in situ signals and counted within layers 2–4. Data (mean ± SD) were averaged across 7–12 sections from at least 3 independent experiments. Scale bar: 100 μm.

Figure 11. Summary of molecularly-defined sSC neuronal cell types

A. Neurons in layer 1 can be divided into two groups: Rorβ+ and Rorβ-. Rorβ+ neurons can be divided again into two groups: Ntng2+ and Ntng2-. Ntng2+ cells can be subdivided into two groups: Cdh7+ and Cdh7-. Ntng2− cells could be also subdivided based on Cdh7 expression, although we could not confirm this classification with our current technique. The combined expression pattern for Ntng2 and Cdh7 divides Rorβ+ neurons into four groups: (1) Ntng2+/Cdh7+, (2) Ntng2+/Cdh7-, (3) Ntng2−/Cdh7+, and (4) Ntng2−/Cdh7-. B. Expression of SST, Brn3b and ETV1 distinguishes subpopulations of neurons in layers 2, 3, and 4. Very few (if any) cells express both SST and Brn3b or ETV1, whereas some cells express both Brn3b and ETV1. Together, neurons in layers 2, 3 and 4 can be subdivided into 5 types: (1) SST+ only, (2) Brn3b+ only, (3) ETV1+ only, (4) both Brn3b+ and ETV1+, and (5) cells negative for all three markers.