Olfactory bulb hypoplasia in Prokr2 null mice stems from defective neuronal progenitor migration and differentiation

Abstract

New neurons are added on a daily basis to the olfactory bulb (OB) of a mammal, and this phenomenon exists throughout its lifetime. These new cells are born in the subventricular zone and migrate to the OB via the rostral migratory stream (RMS). To examine the role of the prokineticin receptor 2 (Prokr2) in neurogenesis, we created a Prokr2 null mouse, and report a decrease in the volume of its OB and also a decrease in the number of bromodeoxyuridine (BrdU)-positive cells. There is disrupted architecture of the OB, with the glomerular layer containing terminal dUTP nick-end labeling (TUNEL) -positive nuclei and also a decrease in tyrosine hydroxylase-positive neurons in this layer. In addition, there are increased numbers of doublecortin-positive neuroblasts in the RMS and increased PSA-NCAM (polysialylated form of the neural cell adhesion molecule) -positive neuronal progenitors around the olfactory ventricle, indicating their detachment from homotypic chains is compromised. Finally, in support of this, Prokr2-deficient cells expanded in vitro as neurospheres are incapable of migrating towards a source of recombinant human prokineticin 2 (PROK2). Together, these findings suggest an important role for Prokr2 in OB neurogenesis.

Fig. 2

TUNEL staining and cell proliferation in the OB. (A) +/+ OB showing lack of TUNEL staining compared with (B) TUNEL-positive staining in the m/m mice shown in higher-power magnification in (C) Note, staining is highest in the GL where architecture is distorted (see also Fig. 1). (D) There is a decrease in bromodeoxyuridine (BrdU) staining in the OB of m/m mice compared with +/+ or +/m 21 days after a single injection of BrdU (***P < 0.001 versus +/+, +/m; n = 6). (E) BrdU staining of OB in +/+ mice (magnification 5 ×). The boxed region in (E) is shown in the corner of (E). (F) BrdU staining in OB of m/m mice (magnification 5 ×), the boxed region in (F) is shown in the corner of (F). Note, the difference in size of the OB at 5 × magnification. Scale bars: 100 µm (A and B); 20 µm (C); 500 µm (E and F).

Fig. 1

Analysis of OBs. Macro- and microscopic analysis of OB from m/m and +/+ mice. (A) Macroscopic view of a m/m mouse compared with a +/+ littermate at 9 weeks old. (B) +/+ OB showing normal size of OB. (C) m/m OB showing decreased size compared with +/+. (D) m/m show decreased volume of the OB compared with either +/+ or +/m (***P < 0.001 versus +/+, +/m; n = 6). (E) Boxed region in (B) and (F), boxed region in (C), comparison of architecture of OB in (B), +/+ vs (C), m/m mice. There is a decrease in the overall size of the OB, which can be contributed to a complete absence of a discernable olfactory nerve layer (ONL), glomerular layer (GL), mitral cell layer (MCL) and internal plexiform layer (IPL). There is evidence of the external plexiform layer (EPL) in the homozygous and also the granule cell layer (GRL). Scale bars: 1 mm (B and C); 200 µm (D and E). Arrows in (E) and (F) indicate that the GRL extends beyond the edge of the figure.

Fig. 3

Neuronal differentiation in olfactory bulb (OB) and RMS. (A) Tyrosine hydroxylase (TH) staining in the GL of the +/+ OB. (B) There is severe depletion of TH in the OB GL of m/m mice compared with either +/+ or +/– (**P < 0.01 versus +/+, +/m; n = 5). (C) DCX-positive neurons in the RMS of +/+. (C′) 20 × magnification. (D) DCX-positive neurons in the RMS of a m/m mouse. (D′) 20 × magnification. Note the greater number of DCX-positive neurons in the RMS of m/m mice, indicating their retention there compared with +/+. (E) PSA-NCAM staining in the olfactory ventricle of +/+, compared with (F), m/m mouse. (G) Higher magnification (20 ×) of (E), (H) higher magnification (20 ×) of (F), demonstrating that PSA-NCAM appears to be in tighter bundles in the m/m compared with +/+ littermates. Scale bars: 10 µm (A); 1 mm (C and D); 250 µm (E and F); 125 µm (G and H).

Fig. 4

Transwell assay. There is a decrease in migration of cells grown from m/m mice towards the Prokr2 receptor ligand PROK2 compared with +/+ or +/m littermates (*P < 0.05 versus Control + m/m, +/m). Control wells contained no PROK2 and demonstrated limited migration over the 24-h period.