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Foxc1 dependent mesenchymal signalling drives embryonic cerebellar growth.

Haldipur P, Gillies GS, Janson OK, Chizhikov VV, Mithal DS, Miller RJ, Millen KJ - Elife (2014)

Bottom Line: In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells.SDF1α also rescues the Foxc1-/- phenotype.Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, United States.

ABSTRACT
Loss of Foxc1 is associated with Dandy-Walker malformation, the most common human cerebellar malformation characterized by cerebellar hypoplasia and an enlarged posterior fossa and fourth ventricle. Although expressed in the mouse posterior fossa mesenchyme, loss of Foxc1 non-autonomously induces a rapid and devastating decrease in embryonic cerebellar ventricular zone radial glial proliferation and concurrent increase in cerebellar neuronal differentiation. Subsequent migration of cerebellar neurons is disrupted, associated with disordered radial glial morphology. In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells. Its receptor, Cxcr4, is expressed in cerebellar radial glial cells and conditional Cxcr4 ablation with Nes-Cre mimics the Foxc1-/- cerebellar phenotype. SDF1α also rescues the Foxc1-/- phenotype. Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

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Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1  mutants.(A–H) Sagittal sections of e13.5 (A, B, E, F) and e15.5 (C, D, G, H) cerebellum from WT (A, C, E, G) and Cxcr4 Conditional Knockout (B, D, F, H) mice. (A–D) Cxcr4 conditional knockout mice showed a reduction in proliferation at e13.5 (B) and e15.5 (D), compared to WT (A, C). Reduced proliferation was accompanied by increased differentiation in the cerebellar ventricular zone of Cxcr4 CKO mice at both e13.5 (F) and e15.5 (H). The ventricular zone in WT cerebellum at both e13.5 (E) and e15.5 (H) largely consisted of β-III Tubulin negative cells. The insets in images (E–H) represent the magnified image of boxed regions within the cerebellar ventricular zone. (I) Graph showing a significant reduction in the percentage of BrdU positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT cerebellar ventricular zone. Data are represented as mean percentage of BrdU positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (J) Graph showing a significant increase in the number of TUNEL positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT animals. Data are represented as mean number of TUNEL positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (K–L) Sagittal sections of e15.5 cerebellum from WT (K) and Cxcr4 CKO (L) mice stained for Calbindin showing aberrant number and position of PCs in the CKO. (K–L) Sagittal sections of e13.5 cerebellum (M, N) and e15.5 (O, P) from WT (M, O) and Cxcr4 CKO (N, P) mice stained for Nestin. The morphology of radial glial fibers in the Cxcr4 CKO greatly resembled that in the Foxc1 mutant. (Q–R) Sagittal sections of e17.5 cerebellum from WT (Q) and Cxcr4 CKO (R) mice stained for BLBP. The morphology and positioning of BG fibers in the Cxcr4 CKO (R, arrows) is similar to the Foxc1 mutant. Abbreviations used include VZ—Ventricular Zone, and Mes—Mesenchyme. Scale bar = 100 µm for all images, except for Q–R where the scale bar = 50 µm.DOI:http://dx.doi.org/10.7554/eLife.03962.010
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fig6: Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1 mutants.(A–H) Sagittal sections of e13.5 (A, B, E, F) and e15.5 (C, D, G, H) cerebellum from WT (A, C, E, G) and Cxcr4 Conditional Knockout (B, D, F, H) mice. (A–D) Cxcr4 conditional knockout mice showed a reduction in proliferation at e13.5 (B) and e15.5 (D), compared to WT (A, C). Reduced proliferation was accompanied by increased differentiation in the cerebellar ventricular zone of Cxcr4 CKO mice at both e13.5 (F) and e15.5 (H). The ventricular zone in WT cerebellum at both e13.5 (E) and e15.5 (H) largely consisted of β-III Tubulin negative cells. The insets in images (E–H) represent the magnified image of boxed regions within the cerebellar ventricular zone. (I) Graph showing a significant reduction in the percentage of BrdU positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT cerebellar ventricular zone. Data are represented as mean percentage of BrdU positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (J) Graph showing a significant increase in the number of TUNEL positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT animals. Data are represented as mean number of TUNEL positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (K–L) Sagittal sections of e15.5 cerebellum from WT (K) and Cxcr4 CKO (L) mice stained for Calbindin showing aberrant number and position of PCs in the CKO. (K–L) Sagittal sections of e13.5 cerebellum (M, N) and e15.5 (O, P) from WT (M, O) and Cxcr4 CKO (N, P) mice stained for Nestin. The morphology of radial glial fibers in the Cxcr4 CKO greatly resembled that in the Foxc1 mutant. (Q–R) Sagittal sections of e17.5 cerebellum from WT (Q) and Cxcr4 CKO (R) mice stained for BLBP. The morphology and positioning of BG fibers in the Cxcr4 CKO (R, arrows) is similar to the Foxc1 mutant. Abbreviations used include VZ—Ventricular Zone, and Mes—Mesenchyme. Scale bar = 100 µm for all images, except for Q–R where the scale bar = 50 µm.DOI:http://dx.doi.org/10.7554/eLife.03962.010

Mentions: As seen in Foxc1−/− mutant cerebella, numbers of BrdU-positive ventricular zone cells were also dramatically reduced in the Cxcr4 CKO at e13.5 and e15.5 (Figure 6A–D,I, Graph). Concurrently, there was an extensive increase in numbers of differentiated β-III tubulin positive cells at e13.5 and e15.5. Similar to Foxc1 mutants, numerous β-III tubulin -positive cells were also ectopically interspersed throughout the diminished Cxcr4 CKO mutant ventricular zone at both stages (Figure 6F,H, Boxed area). Again, similar to Foxc1−/− mutant few calbindin-positive Purkinje cells were present at e15.5 in the Cxcr4 CKO and those remaining were scattered throughout the anlage and located adjacent to the pia (Figure 6L, arrow), instead of remaining in a domain more closely associated with the ventricular zone as in wild-type cerebella (Figure 6K, arrow). Although many nestin-positive radial glial cells were present in the Cxcr4-CKO mutant at e13.5 and e15.5, long radial glial fibers were not present at either stage (Figure 6M–P). Notably, radial glial fibers were still readily detected in the midbrain (Figure 6N, arrows). As seen in the Foxc1−/− mutant, increased cell death was also observed in the Cxcr4 CKO ventricular zone compared to control at e13.5 and e15.5 (Figure 6J). In contrast to the Foxc1−/− mutant cerebellum however, nests of ectopic proliferating granule cell progenitors were present within the Cxcr4-Nes-Cre mutant cerebellar anlage at this stage, as others have previously reported (Figure 6D, asterisk). Bergmann glia were found ectopically located in the EGL similar to the Foxc1−/− mutant (Figure 6R, arrows). We conclude that loss of Cxcr4 in the nestin-expressing radial glial cells recapitulates multiple aspects of the Foxc1−/− mutant cerebellar phenotype. The striking similarities of these mutant cerebellar phenotypes strongly argues that Foxc1-dependent SDF1α secretion by the posterior fossa head mesenchyme and its reception by Cxcr4 in adjacent early cerebellar anlage radial glial cells, represent a major downstream effector pathway mediating Foxc1 Dandy-Walker cerebellar pathogenesis10.7554/eLife.03962.010Figure 6.Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1 mutants.


Foxc1 dependent mesenchymal signalling drives embryonic cerebellar growth.

Haldipur P, Gillies GS, Janson OK, Chizhikov VV, Mithal DS, Miller RJ, Millen KJ - Elife (2014)

Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1  mutants.(A–H) Sagittal sections of e13.5 (A, B, E, F) and e15.5 (C, D, G, H) cerebellum from WT (A, C, E, G) and Cxcr4 Conditional Knockout (B, D, F, H) mice. (A–D) Cxcr4 conditional knockout mice showed a reduction in proliferation at e13.5 (B) and e15.5 (D), compared to WT (A, C). Reduced proliferation was accompanied by increased differentiation in the cerebellar ventricular zone of Cxcr4 CKO mice at both e13.5 (F) and e15.5 (H). The ventricular zone in WT cerebellum at both e13.5 (E) and e15.5 (H) largely consisted of β-III Tubulin negative cells. The insets in images (E–H) represent the magnified image of boxed regions within the cerebellar ventricular zone. (I) Graph showing a significant reduction in the percentage of BrdU positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT cerebellar ventricular zone. Data are represented as mean percentage of BrdU positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (J) Graph showing a significant increase in the number of TUNEL positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT animals. Data are represented as mean number of TUNEL positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (K–L) Sagittal sections of e15.5 cerebellum from WT (K) and Cxcr4 CKO (L) mice stained for Calbindin showing aberrant number and position of PCs in the CKO. (K–L) Sagittal sections of e13.5 cerebellum (M, N) and e15.5 (O, P) from WT (M, O) and Cxcr4 CKO (N, P) mice stained for Nestin. The morphology of radial glial fibers in the Cxcr4 CKO greatly resembled that in the Foxc1 mutant. (Q–R) Sagittal sections of e17.5 cerebellum from WT (Q) and Cxcr4 CKO (R) mice stained for BLBP. The morphology and positioning of BG fibers in the Cxcr4 CKO (R, arrows) is similar to the Foxc1 mutant. Abbreviations used include VZ—Ventricular Zone, and Mes—Mesenchyme. Scale bar = 100 µm for all images, except for Q–R where the scale bar = 50 µm.DOI:http://dx.doi.org/10.7554/eLife.03962.010
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fig6: Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1 mutants.(A–H) Sagittal sections of e13.5 (A, B, E, F) and e15.5 (C, D, G, H) cerebellum from WT (A, C, E, G) and Cxcr4 Conditional Knockout (B, D, F, H) mice. (A–D) Cxcr4 conditional knockout mice showed a reduction in proliferation at e13.5 (B) and e15.5 (D), compared to WT (A, C). Reduced proliferation was accompanied by increased differentiation in the cerebellar ventricular zone of Cxcr4 CKO mice at both e13.5 (F) and e15.5 (H). The ventricular zone in WT cerebellum at both e13.5 (E) and e15.5 (H) largely consisted of β-III Tubulin negative cells. The insets in images (E–H) represent the magnified image of boxed regions within the cerebellar ventricular zone. (I) Graph showing a significant reduction in the percentage of BrdU positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT cerebellar ventricular zone. Data are represented as mean percentage of BrdU positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (J) Graph showing a significant increase in the number of TUNEL positive cells in the cerebellar ventricular zone of Cxcr4 CKO compared to WT animals. Data are represented as mean number of TUNEL positive cells ± s.e.m. *** indicates significance with respect to corresponding WT Control. (p < 0.05). (K–L) Sagittal sections of e15.5 cerebellum from WT (K) and Cxcr4 CKO (L) mice stained for Calbindin showing aberrant number and position of PCs in the CKO. (K–L) Sagittal sections of e13.5 cerebellum (M, N) and e15.5 (O, P) from WT (M, O) and Cxcr4 CKO (N, P) mice stained for Nestin. The morphology of radial glial fibers in the Cxcr4 CKO greatly resembled that in the Foxc1 mutant. (Q–R) Sagittal sections of e17.5 cerebellum from WT (Q) and Cxcr4 CKO (R) mice stained for BLBP. The morphology and positioning of BG fibers in the Cxcr4 CKO (R, arrows) is similar to the Foxc1 mutant. Abbreviations used include VZ—Ventricular Zone, and Mes—Mesenchyme. Scale bar = 100 µm for all images, except for Q–R where the scale bar = 50 µm.DOI:http://dx.doi.org/10.7554/eLife.03962.010
Mentions: As seen in Foxc1−/− mutant cerebella, numbers of BrdU-positive ventricular zone cells were also dramatically reduced in the Cxcr4 CKO at e13.5 and e15.5 (Figure 6A–D,I, Graph). Concurrently, there was an extensive increase in numbers of differentiated β-III tubulin positive cells at e13.5 and e15.5. Similar to Foxc1 mutants, numerous β-III tubulin -positive cells were also ectopically interspersed throughout the diminished Cxcr4 CKO mutant ventricular zone at both stages (Figure 6F,H, Boxed area). Again, similar to Foxc1−/− mutant few calbindin-positive Purkinje cells were present at e15.5 in the Cxcr4 CKO and those remaining were scattered throughout the anlage and located adjacent to the pia (Figure 6L, arrow), instead of remaining in a domain more closely associated with the ventricular zone as in wild-type cerebella (Figure 6K, arrow). Although many nestin-positive radial glial cells were present in the Cxcr4-CKO mutant at e13.5 and e15.5, long radial glial fibers were not present at either stage (Figure 6M–P). Notably, radial glial fibers were still readily detected in the midbrain (Figure 6N, arrows). As seen in the Foxc1−/− mutant, increased cell death was also observed in the Cxcr4 CKO ventricular zone compared to control at e13.5 and e15.5 (Figure 6J). In contrast to the Foxc1−/− mutant cerebellum however, nests of ectopic proliferating granule cell progenitors were present within the Cxcr4-Nes-Cre mutant cerebellar anlage at this stage, as others have previously reported (Figure 6D, asterisk). Bergmann glia were found ectopically located in the EGL similar to the Foxc1−/− mutant (Figure 6R, arrows). We conclude that loss of Cxcr4 in the nestin-expressing radial glial cells recapitulates multiple aspects of the Foxc1−/− mutant cerebellar phenotype. The striking similarities of these mutant cerebellar phenotypes strongly argues that Foxc1-dependent SDF1α secretion by the posterior fossa head mesenchyme and its reception by Cxcr4 in adjacent early cerebellar anlage radial glial cells, represent a major downstream effector pathway mediating Foxc1 Dandy-Walker cerebellar pathogenesis10.7554/eLife.03962.010Figure 6.Aberrations in cell division, differentiation and migration in Cxcr4 radial glial conditional knockout mice are similar to those in Foxc1 mutants.

Bottom Line: In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells.SDF1α also rescues the Foxc1-/- phenotype.Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, United States.

ABSTRACT
Loss of Foxc1 is associated with Dandy-Walker malformation, the most common human cerebellar malformation characterized by cerebellar hypoplasia and an enlarged posterior fossa and fourth ventricle. Although expressed in the mouse posterior fossa mesenchyme, loss of Foxc1 non-autonomously induces a rapid and devastating decrease in embryonic cerebellar ventricular zone radial glial proliferation and concurrent increase in cerebellar neuronal differentiation. Subsequent migration of cerebellar neurons is disrupted, associated with disordered radial glial morphology. In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells. Its receptor, Cxcr4, is expressed in cerebellar radial glial cells and conditional Cxcr4 ablation with Nes-Cre mimics the Foxc1-/- cerebellar phenotype. SDF1α also rescues the Foxc1-/- phenotype. Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

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Related in: MedlinePlus