Transcription Factors Sp8 and Sp9 Coordinately Regulate Olfactory Bulb Interneuron Development

Abstract

Neural stem cells in the postnatal telencephalic ventricular-subventricular zone (V-SVZ) generate new interneurons, which migrate tangentially through the rostral migratory stream (RMS) into the olfactory bulb (OB). The Sp8 and Sp9 transcription factors are expressed in neuroblasts, as well as in the immature and mature interneurons in the V-SVZ-RMS-OB system. Here we show that Sp8 and Sp9 coordinately regulate OB interneuron development: although Sp9 null mutants show no major OB interneuron defect, conditional deletion of both Sp8 and Sp9 resulted in a much more severe reduction of OB interneuron number than that observed in the Sp8 conditional mutant mice, due to defects in neuronal differentiation, tangential and radial migration, and increased cell death in the V-SVZ-RMS-OB system. RNA-Seq and RNA in situ hybridization reveal that, in Sp8/Sp9 double mutant mice, but not in Sp8 or Sp9 single mutant mice, newly born neuroblasts in the V-SVZ-RMS-OB system fail to express Prokr2 and Tshz1 expression, genes with known roles in promoting OB interneuron differentiation and migration, and that are involved in human Kallmann syndrome.

Figure 1.

Sp9 is expressed in the DCX⁺ neuroblasts in the adult V-SVZ. (A) An X-gal stained P30 Sp9^LacZ/+ mouse brain sagittal section showing strong Sp9 expression in the V-SVZ–RMS–OB system. (B–E) The vast majority of DCX⁺ V-SVZ cells expressed Sp9 and vice versa. DCX⁺/Sp9⁺ cells in the lateral (B) and dorsal lateral (C) V-SVZ are shown. (F–J) The numbers of CR⁺, CB⁺, and TH⁺ cells were unaffected in the Sp9-KO OB at P21, except for PV⁺ cells in the EPL, which showed a significant decrease. (Student’s t test, **P < 0.01, n = 3, mean ± SEM). EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; MCL, mitral cell layer. Scale bars: 500 μm in A; 50 μm in B for B, C; 100 μm in I′ for F–I′.

Figure 2.

Severely reduced OB interneurons in Sp8/Sp9-DCKO mice. (A–B′) The OBs and telencephalons from Sp8/Sp9-DCKO mice were smaller than Dlx5/6-CIE controls at P4 and P11. (C–F′) GFP immunostaining and Reln in situ RNA hybridization on P4 and P11 OB sections. Note that most GFP⁺ cells were restricted to the core of the OB in Sp8/Sp9-DCKO mice. (G–H″′) GAD1 and TH in situ RNA hybridization on P11 OB sections of control, Sp9-KO, Sp8-CKO and Sp8/Sp9-DCKO mice. TH mRNA was not detected in Sp8/Sp9-DCKO OBs. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer. Scale bars: 5 mm in B′ for A–B′; 200 μm in D′ for C–D′; 500 μm in H″′ for E–H″′.

Figure 3.

Neural stem/progenitor cells accumulate in the V-SVZ and OB of postnatal Sp8/Sp9-DCKO mice. (A–F′) Representative images of Gsx2⁺ and Ascl1⁺ cells in the V-SVZ–RMS–OB of the control and Sp8/Sp9-DCKO mice at P10. (G, H) The numbers of Gsx2⁺ and Ascl1⁺ cells in the V-SVZ and OB were significantly increased in Sp8/Sp9-DCKO mice compared with controls. (Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3, mean ± SEM). Scale bar: 50 μm.

Figure 4.

Tangential and radial migration defects of neuroblasts in adult hGFAP-Cre; Sp8^F/F; Sp9^F/F mice. (A–D) Whole mount of the lateral wall of the lateral ventricle stained with antibody against DCX showed a marked increase in neuroblast chains in double mutants at P90. (E–L) P90 OB coronal sections stained for NeuN and DCX. Note that hGFAP-Cre; Sp8^F/F; Sp9^F/F double mutant OBs were smaller than control, hGFAP-Cre; Sp9^F/F and hGFAP-Cre; Sp8^F/F OBs. (M–P) High magnification images of DCX⁺ cells in OBs. Arrows indicate defects in radial migration of neuroblasts. R, rostral; C, caudal; D, dorsal; V, ventral. Scale bars: 500 μm in (D) for (A–D); 200 μm in (L) for (E–L); 20 μm in (P) for (M–P).

Figure 5.

Sp8 and Sp9 are required cell autonomously for neuroblast tangential and radial Migration. (A, B) Sp8 and Sp9 were deleted by injecting Ad-Cre into the P0 dorsolateral V-SVZ. GFP⁺ cells in the P7 OB of Rosa-YFP and Sp8^F/F; Sp9^F/F; Rosa-YFP mice were shown. (C, D) GFP⁺ migratory neuroblasts in the P7 RMS. Arrows indicate cells oriented in the direction of the OB, whereas arrowheads indicate cells oriented in the reverse direction. (E) Quantification of the percentage of GFP⁺ cells oriented in the forward direction. (Student’s t test, **P < 0.01, n = 4, mean ± SEM). (F, G) GFP⁺ cells in the P21 OB of Rosa-YFP and Sp8^F/F; Sp9^F/F; Rosa-YFP mice are shown. (H) Quantification of the percentage of GFP⁺ cells that were in the OB core. (Student’s t test, **P < 0.001, n = 3, mean ± SEM). Scale bars: 200 μm in (G) for (A, B, F, G); 50 μm in (D) for (C, D).

Figure 6.

Apoptotic cells are significantly increased in the V-SVZ, RMS and OB in Sp8/Sp9-DCKO mice. (A–F) Cleaved Caspase-3⁺ cells in the V-SVZ–RMS–OB of controls and double mutants at P7. (G–I) Numbers of Caspase-3⁺ cells in the mutant V-SVZ, RMS and OB were significantly higher than those in controls at P4, P7, and P11. (Student’s t-test, ***P < 0.001, n = 3, mean ± SEM). Scale bars: 100 μm in B for A, B; 100 μm in D for C, D; 200 μm in F for E, F.

Figure 7.

Sp8/Sp9-DCKO mice fail to express Prokr2 and Tshz1 in the embryonic dLGE, RMS and OB. (A–F″′) Expression of Prokr2 and Tshz1 were absent in the E16.5 dLGE (arrows), RMS and OB of Sp8/Sp9-DCKO. There were no visible differences in the expression of Tshz1 and Prokr2 in the Sp9-KO compared with controls while in Sp8-CKO was slightly reduced (A″, B″). Note that Tshz1 expression in mitral/tufted cells was unaffected (F″′). Scale bars: 500 μm in B″′ for A–B″′; 200 μm in F″′ for C–F″′.

Figure 8.

Prokr2 and Tshz1 expression in the postnatal V-SVZ, RMS, and OB are dependent on Sp8 and Sp9. (A–I’) Prokr2 and Tshz1 expression in the V-SVZ, RMS, and OB were observed in coronal (A–F″′) and sagittal (G–I′) sections of control, Sp9-KO, Sp8-CKO mice at P4, whereas their expression were lost from these structures in Sp8/Sp9-DCKO mice. Note remaining Tshz1 expression in mitral/tufted cells in the Sp8/Sp9-DCKO OB (F″′, I′). Scale bars: 200 μm in B″′ for A–B″′; 200 μm in D″′ for C–D″′; 500 μm in F″′ for E–F″′; 500 μm in I′ for G–I′.