Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly

Abstract

The vertebrate clustered protocadherin (Pcdh) cell surface proteins are encoded by three closely linked gene clusters (Pcdhα, Pcdhβ, and Pcdhγ). Here, we show that all three gene clusters functionally cooperate to provide individual mouse olfactory sensory neurons (OSNs) with the cell surface diversity required for their assembly into distinct glomeruli in the olfactory bulb. Although deletion of individual Pcdh clusters had subtle phenotypic consequences, the loss of all three clusters (tricluster deletion) led to a severe axonal arborization defect and loss of self-avoidance. By contrast, when endogenous Pcdh diversity is overridden by the expression of a single-tricluster gene repertoire (α and β and γ), OSN axons fail to converge to form glomeruli, likely owing to contact-mediated repulsion between axons expressing identical combinations of Pcdh isoforms.

Fig. 1. Distinct combinations of Pcdhα, -β, and -γ isoforms are stochastically expressed in individual OSNs

Pcdh isoforms are divided into two categories, the alternate (indicated by the yellow, purple, and green ovals) and the C-type (indicated by the blue and red ovals). Single-cell, stochastic expression of Pcdhα and -β and -γ isoforms in five different mOSNs. Note the absence of detectable Pcdhα or -γ c-type expression in these cells (see tables S1 and S2). The presence of individual Pcdh isoform mRNA is indicated by red-colored boxes, and the levels are indicated by the color gradient [log₂ reads per kilobase of transcript per million mapped reads (RPKM)].

Fig. 2. Multiple Pcdh gene clusters are required for normal OSN axonal arborization and the formation of normal protoglomeruli

(A) Immunohistochemistry (IHC) against vesicular glutamate transporter 2 (vglut2) gene expression in coronal sections through the anterior OB of (A) wild-type, (B) Pcdhαβγ^+/−, (C) Pcdhαβγ^−/−, (D) Pcdhα^−/−, (E) Pcdhβ^−/−, and (F) Pcdhγ^−/− pups. Coronal section of the entire anterior bulb (top) and a zoomed-in area (bottom) through the OB. The three Pcdh gene clusters are indicated by the colored boxes. (G) IHC against Venus and vglut2 in utero electroporated OSNs from control and Pcdhαβγ^−/− mice. (H) Quantification of the total length, the number of branch points, and the two-dimensional distribution of OSN arbors in Pcdhαβγ^+/+ (n = 18), Pcdhαβγ^+/− (n = 22), and Pcdhαβγ^−/− mice (n = 32) (n ≥ 6 pups per genotype; Kruskal-Wallis test, P < 0.0001). Error bars represent SEM. DAPI, 4′,6-diamidino-2-phenylindole (blue). All Pcdh-tricluster mutant mice bear also the bacterial artificial chromosome Tg (see fig. S2). Scale bars: (A) to (F), 100 µm; and (G), 20 µm.

Fig. 3. Uni-identity OSN axons fail to form glomeruli

Whole-mount fluorescence microscopy images of the dorsal OB in (A) control and (B and C) uni-identity 4-to 5-week-old mice (mouse strains on figure). Arrows and circles highlight glomeruli in whole-mount zoom images. IHC against GFP, TagT, and β-galactosidase (β-Gal) in a coronal section through the OB in (D) control and (E and F) uni-identity 4- to 5-week-old mice. Dashed line designates the separation of the nerve layer (NL, left side) and the glomerular layer (GL, right side). TagT and β-Gal are pseudo-colored green; DAPI, blue. Scale bars: (A) to (C), 500 µm; (D) to (F), 100 µm.

Fig. 4

Uni-identity like-OSN axons fail to converge in the OB. IHC against β-Gal in coronal sections through the OB in (A) control, (B and C) uni-identity, and (D) UNI ICDΔ in 4-week-old mice. Arrows depict the P2 medial glomerulus. Dashed line designates the separation of NL (right) and GL (left). β-Gal is pseudo-colored green. (E) Quantification of P2 axonal distribution of lateral and medial projections in the OB of 8-week-old control (black, n = 9 bulbs), and uni-identity mice (red, n = 6 bulbs). (Mann-Whitney test, medial P = 0.0002, lateral P = 0.0004). (F) Normally, like-OSN axons (OR1, red, and OR2, green) converge into stereotypically positioned glomeruli within the OB (left). OE, olfactory epithelium. In uni-identity mice, OSN axons that share the same single dominant Pcdh uni-identity project diffusely to their expected positions in the OB (right). (G) Expression of GFP or (H) UNI3, or (I) UNI ICDΔ cassette exclusively in MOR28 OSNs. Arrows depict the aberrant projection of MOR28-UNI axons in the bulb. [J (a to c)] The few MOR28-UNI axons localize with wild-type MOR28 OSNs labeled with GFP. Animals were 4 to 5 weeks old. DAPI, blue. Scale bar, 100 µm.