1. Neuroscience

Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice

  1. Hongsheng Zhang
  2. Anupama Sathyamurthy
  3. Fang Liu
  4. Lei Li
  5. Lei Zhang
  6. Zhaoqi Dong
  7. Wanpeng Cui
  8. Xiangdong Sun
  9. Kai Zhao
  10. Hongsheng Wang
  11. Hsin-Yi Henry Ho
  12. Wen-Cheng Xiong
  13. Lin Mei  Is a corresponding author
  1. Case Western Reserve University, United States
  2. Augusta University, United States
  3. Harvard Medical School, United States
  4. Louis Stokes Cleveland Veterans Affairs Medical Center, United States
https://doi.org/10.7554/eLife.45303
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Cite this article
  1. Hongsheng Zhang
  2. Anupama Sathyamurthy
  3. Fang Liu
  4. Lei Li
  5. Lei Zhang
  6. Zhaoqi Dong
  7. Wanpeng Cui
  8. Xiangdong Sun
  9. Kai Zhao
  10. Hongsheng Wang
  11. Hsin-Yi Henry Ho
  12. Wen-Cheng Xiong
  13. Lin Mei
(2019)
Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice
eLife 8:e45303.
https://doi.org/10.7554/eLife.45303
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6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Requirement of Agrin for adult hippocampal neurogenesis.

(A) Schematic diagram of standard cage (SC) and environmental enrichment (EE) housing. (B–C) Increased Arc+ cells in hippocampus of mice in EE, compared with SC-housed mice. n = 3 for each group, Student’s t-test: t (4)=5.493, p=0.0054. (D) Increased Agrn mRNA level in hippocampus of EE-housed mice, compared with SC-housed mice. n = 3 for each group. Student’s t-test: t (4)=3.641, p=0.022 (Bdnf); t (4)=9.545, p=0.0007 (Igf1); t (4)=3.32, p=0.0294 (Vegf); t (4)=3.434, p=0.0264 (Agrn); t (4)=0.7758, p=0.4812 (ApoE); t (4)=0.3968, p=0.7117 (Wnt5a). (E) Increased Lrp4 mRNA level in hippocampus of EE-housed mice, compared with SC-housed mice. n = 3 for each group. Student’s t-test: t (4)=8.04, p=0.0013 (Lrp4); t (4)=1.76, p=0.1527(MuSK). (F–I) Reduced BrdU, Mcm2, and Dcx-labeled cells in Agrin CKO hippocampal SGZ. (F) Representative images. Scale bar,100 µm. (G–I) Stereological quantification of BrdU+ (G), Mcm2+ (H), and Dcx+ (I) cells. n = 4 for each group. Student’s t-test: t (6)=3.656, p=0.0106 for BrdU; t (6)=4.185, p=0.0058 for Mcm2; t (6)=3.410, p=0.0143 for Dcx. (J) Agrin CKO mice increased latency to find the hidden platform F(1,70)=7.81, p=0.0067. (K) Reduced time spent in target quadrant. n = 8 for each group, Student’s t test: t (14)=2.639, p=0.0195. (L) Reduced number of platform crossings. n = 8 for each group, Student’s t test: t (14)=0.0386. (M) Reduced preference score during test section. n = 8 for each group, Student’s t test t (14)=2.865, p=0.0125. (N–O) Increased immobility of Agrin CKO mice, compared with control mice, in FST (N) and TST (O). n = 8 for each group. Student’s t-test: t (14)=3.956, p=0.0014 for FST; t (14)=2.691, p=0.0175 for TST. Data are mean ± s.e.m; *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.002
Figure 1—source data 1

Requirement of Agrin for adult hippocampal neurogenesis.

https://doi.org/10.7554/eLife.45303.004
Figure 1—source data 2

Characterization of neuronal Agrn knockout mice.

https://doi.org/10.7554/eLife.45303.005
Figure 1—figure supplement 1
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Generation and characterization of neuronal Agrn knockout mice.

(A) Agrnf/f mice were crossed with Neurod6-Cre mice. Resulting Neurod6-Cre::Agrnf/+ mice were crossed with Agrnf/+ mice to generate Neurod6-Cre::Agrnf/f (Agrin CKO) and Agrnf/+ or Agrnf/f (Control). (B) Reduced Agrn mRNA levels in Agrin CKO hippocampus, compared with control mice. nAgrn, neuronal Agrn. n = 3 for each group. Student’s t-test: t (4)=12.5, p=0.0002 (total Agrn); t (4)=28.1, p<0.001 (nAgrn). (C) Similar body weights between control and Agrin CKO mice. n = 8 for each group, P56. Student’s t-test: t (14)=0.4794, p=0.6391. (D) Similar hippocampal morphology between control and Agrin CKO mice. Scale bar, 200 µm. (E) Similar hippocampus volume between control and Agrin CKO mice at P60. Student’s t-test: t (4)=0.4905, p=0.6495. (F) Agrin CKO shows similar swim speed with control in water maze; t (14)=0.07001, p=0.9452. (G) Similar preference score during training section. n = 8 for each group, Student’s t test t (14)=0.5091, p=0.6186. Data are mean ± s.e.m; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.003
Figure 2
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Lrp4 expression in adult hippocampal NSPCs in mice.

(A) X-gal staining of coronal brain sections of Lrp4-LacZ mice. Arrowheads, astrocytes; arrows, NSPCs. Scale bar, 100 µm. (B) Lrp4 expression in neural stem cells labeled by Gfap and Nestin. DG sections of Lrp4-LacZ mice were stained for β-gal, Gfap, Nestin, and DAPI. A representative cell was circled that was positive for Gfap and Nestin. Scale bar, 5 µm. (C, D) No-detectable β-gal level in Tbr2+ (C) and PSA-NCAM (D) cells in DG. Scale bar, 5 µm.

https://doi.org/10.7554/eLife.45303.006
Figure 3 with 2 supplements
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Reduced NSPCs proliferation and increased immobility of Lrp4 CKO mice.

(A–C) Reduced numbers of BrdU- and Dcx-labeled cells in Lrp4 CKO SGZ. (A) Representative images. Scale bar, 100 μm. (B–C) Stereological quantification of SGZ BrdU+ (B) and Dcx+ (C) cells. n = 5 for each group. Student’s t-test: t (8)=6.602, p=0.0002 for BrdU; t (8)=4.701, p=0.0015 for Dcx. (D–F) Reduced NPCs proliferation in Lrp4 CKO. (D) Representative images. The arrow indicated Gfap+/BrdU+/Sox2+, while the arrow head indicated BrdU+/Sox2+ cells. Scale bar, left 100 µm, right 20 µm. (E) Similar numbers of SGZ Gfap+Sox2+ BrdU+ NSCs between the two genotypes. n = 5 for each group. Student’s t-test: t (8)=0.2947, p=0.7757. (F) Decreased the density of Sox2+BrdU+ NPCs in Lrp4 CKO mice, compared with control. n = 5 for each group. Student’s t-test: t (8)=7.943, p<0.0001. (G–H) Increased duration of immobility in FST (G) and TST (H) of Lrp4 CKO mice, compared with control. n = 8 for each group. Student’s t-test: t (14)=2.826, p=0.0135 for FST; t (14)=2.332, p=0.0352 for TST. Data are mean ± s.e.m; *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.007
Figure 3—source data 1

Reduced adult neurogenesis and increased immobility of Lrp4 CKO mice.

https://doi.org/10.7554/eLife.45303.010
Figure 3—source data 2

Characterization of Lrp4 CKO mice.

https://doi.org/10.7554/eLife.45303.011
Figure 3—source data 3

Similar number of cleaved caspase-3 labeled cells between Lrp4 CKO and control mice.

https://doi.org/10.7554/eLife.45303.012
Figure 3—figure supplement 1
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Generation and characterization of Lrp4 mutant mice.

(A) Lrp4f/f mice were crossed with hGFAP-Cre mice; resulting GFAP-Cre::Lrp4f/+ were crossed with Lrp4f/f mice to generate hGFAP-Cre::Lrp4f/f (Lrp4 CKO) and Lrp4f/+ or Lrp4f/f (control). (B) Reduced Lrp4 mRNA in Lrp4 CKO hippocampus, compared with control. n = 3 for each group. Student’s t-test: t (4)=15.76, p<0.0001. (C–D) Reduced Lrp4 protein in Lrp4 CKO hippocampus, compared with control. n = 3 for each group. Student’s t-test: t (4)=10.49, p=0.0005. Data are mean ± s.e.m. ***, p<0.001.

https://doi.org/10.7554/eLife.45303.008
Figure 3—figure supplement 2
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Similar number of cleaved caspase-3 labeled cells between Lrp4 CKO and control mice.

(A) Representative images. Scale bar, left 50 µm, right 5 µm. (B) Quantitative data. n = 3 for each group. Student’s t-test: t (4)=0.2427, p=0.8202. Data are mean ± s.e.m; ns, p>0.05.

https://doi.org/10.7554/eLife.45303.009
Figure 4 with 2 supplements
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Cell-autonomous effect of Lrp4 in regulating NSPCs proliferation and behavior improvement.

(A) The protocol of Tam treatment. (B–C) Decreased Td+ cells in Nes Lrp4 CKO mice compared with control at 2 days after Tam treatment. (B) Representative images. Scale bar, 25 µm. (C) Stereological quantification of Td+ cell density. n = 4 for each group. Student’s t-test: t (6)=6.553, p=0.0006. (D–E) Decreased Td+ cells in Nes Lrp4 CKO mice compared with control after 1 months of Tam treatment. (D) Representative images. Scale bar, 50 µm. (E) Stereological quantification of Td+ cell density. Student’s t-test: t (4)=8.159, p=0.0012. (F) Time schedule of Tam injection, EE, and BrdU administration. (G–H) EE for 4 weeks failed to increase the density of Ki67+ cells in the DG of iNestin-Lrp4f/f mice. (G) Representative images, Scale bar, 100 µm. (H) Stereological quantification of Ki67+ cell density. n = 3 for each group. Two-way ANOVA test, F (1,8)=129.4, p<0.0001 for genotype; F(1,8) = 12.4, p=0.0078 for EE). (I–L) EE for 4 weeks failed to increase the density of BrdU+, Gfap+Sox2+BrdU+, Sox2+BrdU+ cells in the DG of Nes Lrp4 CKO mice. (I) Representative images, Scale bar, 100 µm. (J) EE for 4 weeks failed to increase the density of BrdU+ cells in the DG of Nes Lrp4 CKO mice. n = 3 for each group. Two-way ANOVA test, F (1,8)=57.01, p<0.0001 for genotype; F (1,8)=24.28, p=0.0012 for EE. (K) EE for 4 weeks increase the density of Gfap+Sox2+BrdU+ cells in the DG of Nes Lrp4 CKO mice. n = 3 for each group. Two-way ANOVA test, F (1,8)=10.09, p=0.0131 for genotype; F (1,8)=8.321, p=0.0204 for EE. (L) EE for 4 weeks increase the density of Sox2+BrdU+ cells in the DG of Nes Lrp4 CKO mice. n = 3 for each group. Two-way ANOVA test, F (1,8)=98.21, p<0.0001 for genotype; F (1,8)=14.09, p=0.0056 for EE. (M–N) Nes Lrp4 CKOmice did not display decrease the duration of immobility in FST (K) and TST (L) after EE for 4 weeks. n = 6 for each group. In FST, two-way ANOVA test: F (1,20)=115.5, p<0.0001 for genotype; F (1,20)=9.04, p=0.007 for EE. In TST, two-way ANOVA test: F (1,20)=44.99, p<0.0001 for genotype; F (1,20)=11.85, p=0.0026 for EE. Data are mean ± s.e.m; ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.013
Figure 4—source data 1

Cell-autonomous effect of Lrp4 in regulating NSPCs proliferation and behavior improvement.

https://doi.org/10.7554/eLife.45303.016
Figure 4—source data 2

Characterization of inducible NSPCs-specific Lrp4 knockout mice.

https://doi.org/10.7554/eLife.45303.017
Figure 4—source data 3

Impaired maturation of Lrp4 mutant newborn neurons.

https://doi.org/10.7554/eLife.45303.018
Figure 4—figure supplement 1
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Generation and characterization of inducible NSPCs-specific Lrp4 knockout mice.

(A) Strategy of mouse crossing and Tam treatment. (B) Tam treatment diagram. (C) Decreased Lrp4 mRNA in the DG of Nes Lrp4 CKO mice after Tam treatment. n = 3 for each group. Student’s t-test: t (4)=15.18, p=0.0001. (D–E) Reduced Lrp4 protein in Nes Lrp4 CKO DG after Tam treatment. n = 3 for each group. Student’s t-test: t (4)=6.31, p=0.0032. (F–G) Similar number of cleaved Caspase-3 labeled cells between Nes Lrp4 CKO and control mice 2 days after last Tam treatment. (F) Representative images, Scale bar 20 µm. (G) Quantitative data, n = 4 for each group. Student’s t test: t (6)=0.7717, p=0.4696. Data are mean ± s.e.m; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.014
Figure 4—figure supplement 2
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Impaired maturation of Lrp4 mutant newborn neurons.

(A) Schematic diagram of retroviral injection. (B–F) Impaired dendrite maturation of Lrp4 mutant newborn neurons. (B) Representative image. Scale bar, 50 µm. (C) Decreased numbers of dendrite branches of GFP+ newborn neurons that expressed Cre, compared with those that expressed D-Cre. dpi, days post injection. n = 36 for D-Cre 28 dpi; n = 42 for Cre 28 dpi; n = 31 for D-Cre 42 dpi; n = 29 for Cre 42 dpi. Student’s t test: t (76)=5.234, p<0.0001 for 28 dpi; t (58)=3.094, p=0.003 for 42 dpi. (D) Reduced total dendrite length of GFP+ newborn neurons that expressed Cre, compared with those that expressed D-Cre. n = 36 for D-Cre 28 dpi; n = 42 for Cre 28 dpi; n = 31 for D-Cre 42 dpi; n = 29 for Cre 42 dpi. Student’s t-test: t (76)=2.974, p=0.0039 for 28 dpi; t (58)=4.332, p<0.0001 for 42 dpi. (E–F) Decreased dendrite complexity by Lrp4 mutation. Two-way ANOVA followed by Bonferroni multiple comparisons test, F (1,1949)=92.61, p<0.0001 for 28 dpi (E); F(1,1450) = 64.91, p<0.0001 for 42 dpi (F). (G–H) Impaired dendritic spine development. (G and H) Representative images. Scale bar, 2 µm. (I) Decreased spine density in Lrp4 mutant newborn neurons. n = 26 for D-Cre 28 dpi; n = 26 for Cre 28 dpi; n = 26 for D-Cre 42 dpi; n = 26 for Cre 42 dpi. Two-way ANOVA with Tukey’s multiple comparisons test, F (1,100)=37.42, p<0.0001 for 28 and 42 dpi. F (1,100)=130.1, p<0.0001 for D-Cre and Cre. p<0.0001 for D-Cre 28 dpi and Cre 28 dpi. p<0.0001 for D-Cre 28 dpi and 42 D-Cre dpi. p<0.0001 for D-Cre 42 dpi and Cre 42 dpi. p=0.0049 for Cre 28 dpi and Cre 42 dpi. More than three mice were analyzed in each genotype for 28 dpi and 42 dpi. Data are mean ± s.e.m. **, p<0.01, ***, p<0.001.

https://doi.org/10.7554/eLife.45303.015
Figure 5 with 1 supplement
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Requirement of the β1 propeller domain for Lrp4 regulation of adult neurogenesis.

(A) Domain structures of Lrp4 and deletion mutants. (B–C) Reduced Dcx+ cell density in Lrp4 CKO and Lrp4 CKO ∆β1, compared with control and Lrp4 CKO ∆β3 mice. (B) Representative images. Scale bar, 50 µm. (C) Stereological quantification of Dcx+ cell density. n = 3 for each group. One-way ANOVA multiple comparisons test, F (3,8)=18.69, p=0.0006; control vs Lrp4 CKO, p=0.0011; control vs Lrp4 CKO ∆β1, p=0.0012; control vs Lrp4 CKO ∆β3, p=0.7005. (D–E) Reduced BrdU+ cell density in Lrp4 CKO and Lrp4 CKO ∆β1, compared with control and Lrp4 CKO ∆β3 mice. (D) Representative images. Scale bar, 50 µm. (E) Stereological quantification. One-way ANOVA multiple comparisons test, F (3,8)=21.26, p=0.0004; control vs Lrp4 CKO, p=0.0005; control vs Lrp4 CKO ∆β1, p=0.0011; control vs Lrp4 CKO ∆β3, p=0.5538. Data are mean ± s.e.m. ns, p>0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.019
Figure 5—source data 1

Requirement of the β1 propeller domain for Lrp4 regulation of adult neurogenesis.

https://doi.org/10.7554/eLife.45303.021
Figure 5—figure supplement 1
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Generation of Lrp4 CKO ∆β1 and Lrp4 CKO ∆β3 mice.

(A) Transgene constructs. (B) Mouse crossing strategy. mt indicates either Lrp4-∆β1 or Lrp4-∆β3. (C–D) Genotyping of indicated mice. wt allele generated 415 bp; floxed allele generated 455 bp; LSL-∆β1 generated ~567 bp; LSL-∆β3 generated ~842 bp; and GFAP-Cre generated ~150 bp bands. (E) Expression of Lrp4-∆β1 and Lrp4-∆β3 in indicated mouse strains, n = 3 for each group. Western blotting was performed with indicated antibodies.

https://doi.org/10.7554/eLife.45303.020
Figure 6 with 2 supplements
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Requirement of Ror2 for adult neurogenesis.

(A–B) Co-immunoprecipitation Ror2 (A), not Ror1 (B), with Lrp4 in co-transfected HEK293T cell. (C–D) Increased Lrp4-Ror2 interaction in Agrin-treated HEK293T cell. (D) Quantitative analysis of data of C. Lrp4 intensity was normalized by that of IgG. Student’s t-test: t (4)=18.47, p<0.0001. (E) Increased Ror2 tyrosine phosphorylation in Agrin-treated neurosphere. Three independent experiments were performed. (F–G) Increased neurosphere size by Agrin and blockade by Lrp4 or Ror2 mutation. (F) Representative images. Scale bar, 100 µm. (G) Quantification of neurosphere size. One-way ANOVA test: F (5,314)=28.55, p<0.0001. Three independent experiments were performed. (H–I) Decreased Dcx+ cell density in Nes Ror2 CKO mice, compared with control. (H) Representative images. Scale bar, 100 µm. (I) Stereological quantification of Dcx+ cell density. n = 5 for each group. Student’s t-test: t (8)=4.523, p=0.0019. (J–L) Reduced BrdU+ cell density in Nes Ror2 CKO mice, compared with control. (J) Representative images. Scale bar 100 µm. (K) Stereological quantification of BrdU+ cell density. n = 5 for each group. Student’s t-test: t (8)=5.948, p=0.0003. (L) Similar density of SGZ Gfap+Sox2+ BrdU+ NSCs between the two genotypes. Student’s t-test: t (8)=1.22, p=0.2572. Data are mean ± s.e.m. **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.022
Figure 6—source data 1

Requirement of Ror2 for adult neurogenesis.

https://doi.org/10.7554/eLife.45303.025
Figure 6—source data 2

Characterization of Ror2 mutant mice.

https://doi.org/10.7554/eLife.45303.026
Figure 6—source data 3

Characterization of inducible NSPCs-specific Ror2 knockout mice.

https://doi.org/10.7554/eLife.45303.027
Figure 6—figure supplement 1
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Generation and characterization of Ror2 mutant mice.

(A) Generation of Ror2 conditional knockout mice. (B–C) Reduced Ror2 protein in Ror2 CKO hippocampus, compared with control. (B) Representative western blot. (C) Quantitative data of B. n = 3 for each group. Student’s t-test: t (4)=22.97, p<0.0001. (D) Similar brain gross morphology of Ror2 CKO and control mice. Scale bar, 5 mm. (E) Similar hippocampus morphology between control and Ror2 CKO mice. (F) Similar hippocampus volume of Ror2 CKO and control mice at P60. Student’s t-test: t (4)=0.416, p=0.6988. Scale bar, 200 µm. (G–H) Reduced density of Dcx+ cells in Ror2 CKO DG, compared with control. (G) Representative images. Scale bar, 50 µm. (H) Stereological. n = 4 for control and n = 3 for Ror2 CKO. Student’s t-test: t (5)=3.77, p=0.013. (I–J) Decreased numbers of BrdU+ cells in Ror2 CKO DG, compared with control. (I) Representative images. Scale bar 50 µm. (J) Stereological quantification. n = 4 for control and n = 3 for Ror2 CKO. Student’s t-test: t (5)=3.538, p=0.0166. Data are mean ± s.e.m. *, p<0.05; ***, p<0.001.

https://doi.org/10.7554/eLife.45303.023
Figure 6—figure supplement 2
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Generation and characterization of inducible NSPCs-specific Ror2 knockout mice.

(A) Strategy of mouse crossing and Tam treatment. (B) Tam treatment diagram. (C) Reduced Ror2 protein in Nes Ror2 CKO DG after Tam treatment. n = 3 for each group. Student’s t-test: t (4)=7.7, p=0.0015. Data are mean ± s.e.m; **, p<0.01.

https://doi.org/10.7554/eLife.45303.024

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(M. musculus)		AgrnfJackson LaboratoryStock #: 031788Harvey et al., 2007
Genetic reagent
(M. musculus)		Lrp4fWu et al., 2012
Genetic reagent
(M. musculus)		Ror2fJackson LaboratoryStock #: 018354Ho et al., 2012
Genetic reagent
(M. musculus)		Neurod6-CreCARD R-BASECARD ID: 2556Goebbels et al., 2006
Genetic reagent
(M. musculus)		GFAP-CreJackson
Laboratory		Stock #: 004600Zhuo et al., 2001
Genetic reagent
(M. musculus)		Ai9 (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J)Jackson LaboratoryStock #: 007909Madisen et al., 2010
Genetic reagent
(M. musculus)		Nes-Cre/ERT2 (C57BL/6Tg(Nes-cre/ERT2)KEisc/J)Jackson LaboratoryStock #: 016261PMID:17166924
Genetic reagent
(M. musculus)		Lrp4-LacZKNOCKOUT MOUSE PROJECTProject ID: VG15248Sun et al., 2018
Genetic reagent
(M. musculus)		LSL-Lrp4-Δβ1This paper
Genetic reagent
(M. musculus)		LSL-Lrp4-Δβ3This paper
Cell line (Homo sapiens)HEK293TATCCCat#:CRL-3216
RRID: CVCL_0042
Cell line (Homo sapiens)GP2-293ClontechCat #: 631458
RRID: CVCL_WI48
AntibodyMouse anti-ArcSanta Cruz BiotechnologyCat #: sc-7839
RRID: AB_626696		IHC (1:200)
AntibodyGoat anti-DcxSanta Cruz BiotechnologyCat #: sc-8066
RRID: AB_2088494		IHC (1:200)
AntibodyMouse anti-Mcm2BD BiosciencesCat #: 610701
RRID: AB_398024,		IHC (1:500)
AntibodyRat anti-BrdUAccurate Chemical
and Scientific Corporation		Cat #: OBT0030
RRID: AB_2313756		IHC (1:500)
AntibodyRabbit anti-Ki67MilliporeCat #: AB9260
RRID: AB_2142366		IHC (1:200)
AntibodyMouse anti-NestinBD BiosciencesCat #: 556309
RRID: AB_396354		IHC (1:200)
AntibodyRabbit anti-GFAPDakoCat #: Z0334
RRID: AB_10013382		IHC (1:1000)
AntibodyChicken anti-β-galAves LabsCat #: BGL-1040
RRID: AB_2313507		IHC (1:1000)
AntibodyMouse anti-Sox2Santa Cruz BiotechnologyCat #: sc-20088
RRID: AB_2255358		IHC (1:200)
AntibodyRabbit anti-Tbr2AbcamCat #: ab23345
RRID: AB_778267		IHC (1:1000)
AntibodyMouse anti-PSA-NCAMMilliporeCat #: MAB5324
RRID: AB_95211		IHC (1:500)
AntibodyRabbit anti-Cleaved Caspase3Cell Signaling TechnologyCat #: 9661
RRID: AB_2341188		IHC (1:200)
AntibodyMouse anti-NeuNMilliporeCat #: MAB377
RRID: AB_2298772		IHC (1:1000)
AntibodyChicken anti-GFPAVESCat #: GFP-1020
RRID: AB_10000240		IHC (1:1000)
AntibodyRabbit anti-FlagSigma-AldrichCat #: F7425
RRID: AB_439687		WB (1:1000)
AntibodyMouse anti-HASigma-AldrichCat #: H9658
RRID: AB_260092		WB (1:5000)
AntibodyMouse anti-GAPDHSanta Cruz
Biotechnology		Cat #: sc-32233, RRID: AB_627679WB (1:10000)
AntibodyMouse anti-β-ActinCell Signaling TechnologyCat #: 12262
RRID: AB_2566811		WB (1:5000)
AntibodyMouse anti-P-Tyr-100Cell Signaling
Technology		Cat #: 9411
RRID: AB_331228		WB (1:1000)
AntibodyRabbit anti-Ror2Cell Signaling TechnologyCat #: 4105
RRID: AB_2180134		WB (1:1000)
AntibodyMouse anti-Lrp4UC Davis/NIH
NeuroMab Facility		Cat #: 75–221
RRID: AB_2139030		WB (1:1000)
AntibodyAlexa Fluor 647-AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H + L)Jackson Immuno
Research Labs		Cat #: 711-607-003 RRID: AB_2340626IHC (1:200)
AntibodyAlexa Fluor
594-AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H + L)		Jackson Immuno
Research Labs		Cat #: 711-586-152 RRID: AB_2340622IHC (1:200)
AntibodyAlexa Fluor 488-AffiniPure Fab
Fragment Donkey Anti-Rabbit IgG (H + L)		Jackson ImmunoResearch LabsCat #: 711-547-003 RRID: AB_2340620IHC (1:200)
AntibodyAlexa Fluor 647-AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H + L)Jackson ImmunoResearch LabsCat #: 715-607-003 RRID: AB_2340867IHC (1:200)
AntibodyAlexa Fluor 488-AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H + L)Jackson ImmunoResearch LabsCat #: 715-547-003
RRID: AB_2340851		IHC (1:200)
AntibodyAlexa Fluor 488-AffiniPure Fab Fragment Donkey Anti-Goat IgG (H + L)Jackson ImmunoResearch LabsCat #: 705-547-003 RRID: AB_2340431IHC (1:200)
AntibodyAlexa Fluor
488-AffiniPure
F(ab')2 Fragment
Donkey Anti-Chicken IgY (IgG) (H + L)		Jackson ImmunoResearch LabsCat #: 703-546-155 RRID: AB_2340376IHC (1:200)
AntibodyAlexa Fluor 647-
AffiniPure Fab Fragment Donkey Anti-Rat IgG (H + L)		Jackson ImmunoResearch LabsCat #: 712-607-003, RRID: AB_2340697IHC (1:200)
AntibodyIRDye 680RD Donkey anti-Rabbit IgG (H + L)LI-COR BiosciencesCat #: 926–68073, RRID:AB_10954442WB (1:10000)
AntibodyDonkey Anti-Mouse IgG, IRDye 800CW ConjugatedLI-COR BiosciencesCat # 926–32212, RRID: AB_621847WB (1:10000)
Recombinant DNA reagentpFlag-Lrp4PMID: 30171091Materials and methods subsection: antibodies and plasmid
Recombinant
DNA reagent		HA-Ror1This paperMaterials and methods subsection: antibodies and plasmid
Recombinant DNA reagentHA-Ror2This paperMaterials and methods subsection: antibodies and plasmid
Chemical compound,drugBrdUSigmaCat #: B5002
Chemical compound,drugTamoxifenSigmaCat #: T5648
Software, algorithmImage JNIH, USARRID:SCR_003070

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https://doi.org/10.7554/eLife.45303.028

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  1. Hongsheng Zhang
  2. Anupama Sathyamurthy
  3. Fang Liu
  4. Lei Li
  5. Lei Zhang
  6. Zhaoqi Dong
  7. Wanpeng Cui
  8. Xiangdong Sun
  9. Kai Zhao
  10. Hongsheng Wang
  11. Hsin-Yi Henry Ho
  12. Wen-Cheng Xiong
  13. Lin Mei
(2019)
Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice
eLife 8:e45303.
https://doi.org/10.7554/eLife.45303