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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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SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.(A) Schematic of dorsal whole mount view of the embryonic mouse head and a brief description of experiments included in the figure. (B) Graph showing the percentage of BrdU+ cells in primary dissociated cerebellar culture. Addition of SDF1α significantly increased BrdU uptake by cells. Addition of AMD3100, an antagonist of SDF1α significantly reduced BrdU incorporation. (C, D; C′, D′) Sagittal sections of e13.5 cerebellum cultured whole mount for 1 day and stained for Ki67 (inset) and β-III Tubulin. Addition of AMD3100 to the culture significantly reduced cell proliferation and increased differentiation in the ventricular zone (D). (E) Graph showing the percentage of Ki67 and (F) β-III Tubulin+ cells in the cerebellar ventricular zone. (G) Schematic describing the transwell migration assay. Addition of SDF1α significantly increased the number of cells migrating through the membrane of the insert. Addition of AMD3100 to the upper well significantly lowered the number of migrating cells (H). (H) Graph quantifying the results of the experiment described in (G) (I) Schematic of a sagittal section of the e13.5 cerebellum. Ventricular zone progenitors are labelled in yellow, while Ptf1a positive cells that exit the ventricular zone are marked red. RL progenitors are labelled light green while granule cell progenitors exiting the RL are labelled dark green. (J–K′′) Matrigel assay to study the effect of SDF1α on the neuronal migration. Ptf1a positive cells from an e13.5 cerebellar slice, when incubated with SDF1α coated acrylic beads were seen to move towards the source of the chemokine (K–K′′). Saline coated beads had no effect on migration (J, J′). Abbreviations used; VZ—Ventricular Zone, RL—Rhombic Lip, EGL—External Granular Layer and Mes—Mesenchyme. The white dotted line in (C–D′) represents the cerebellar ventricular surface. Scale bar = 100 µm. In Graphs (B), (E, F) and (H), data is represented as mean percentage of BrdU or β-III Tubulin positive cells or migrating cells ± s.e.m. In (B) and (H) ** indicates significance with respect to Control, while *** indicates significance with respect to SDF1α treatment. In (E, F) *** indicates significance with respect to Control. (p < 0.05), for all data.DOI:http://dx.doi.org/10.7554/eLife.03962.008
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fig4: SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.(A) Schematic of dorsal whole mount view of the embryonic mouse head and a brief description of experiments included in the figure. (B) Graph showing the percentage of BrdU+ cells in primary dissociated cerebellar culture. Addition of SDF1α significantly increased BrdU uptake by cells. Addition of AMD3100, an antagonist of SDF1α significantly reduced BrdU incorporation. (C, D; C′, D′) Sagittal sections of e13.5 cerebellum cultured whole mount for 1 day and stained for Ki67 (inset) and β-III Tubulin. Addition of AMD3100 to the culture significantly reduced cell proliferation and increased differentiation in the ventricular zone (D). (E) Graph showing the percentage of Ki67 and (F) β-III Tubulin+ cells in the cerebellar ventricular zone. (G) Schematic describing the transwell migration assay. Addition of SDF1α significantly increased the number of cells migrating through the membrane of the insert. Addition of AMD3100 to the upper well significantly lowered the number of migrating cells (H). (H) Graph quantifying the results of the experiment described in (G) (I) Schematic of a sagittal section of the e13.5 cerebellum. Ventricular zone progenitors are labelled in yellow, while Ptf1a positive cells that exit the ventricular zone are marked red. RL progenitors are labelled light green while granule cell progenitors exiting the RL are labelled dark green. (J–K′′) Matrigel assay to study the effect of SDF1α on the neuronal migration. Ptf1a positive cells from an e13.5 cerebellar slice, when incubated with SDF1α coated acrylic beads were seen to move towards the source of the chemokine (K–K′′). Saline coated beads had no effect on migration (J, J′). Abbreviations used; VZ—Ventricular Zone, RL—Rhombic Lip, EGL—External Granular Layer and Mes—Mesenchyme. The white dotted line in (C–D′) represents the cerebellar ventricular surface. Scale bar = 100 µm. In Graphs (B), (E, F) and (H), data is represented as mean percentage of BrdU or β-III Tubulin positive cells or migrating cells ± s.e.m. In (B) and (H) ** indicates significance with respect to Control, while *** indicates significance with respect to SDF1α treatment. In (E, F) *** indicates significance with respect to Control. (p < 0.05), for all data.DOI:http://dx.doi.org/10.7554/eLife.03962.008

Mentions: We hypothesized that Foxc1 regulates the expression of secreted factors in the head mesenchyme which then non-autonomously influence the developing cerebellum. Based on candidate gene analysis, we previously reported that Foxc1−/− mutant posterior fossa mesenchyme has reduced expression of SDF1α (Cxcl12), Bmp2 and 4 (Aldinger et al., 2009). We tested the ability of these secreted factors to influence cerebellar ventricular zone proliferation and migration of ventricular zone-derived neurons using various assays (Figure 4). The embryonic cerebellar ventricular zone does not express Bmp-receptors although they are expressed in the rhombic lip (Machold et al., 2007). In contrast, the SDF1α receptor, Cxcr4 is highly expressed in the embryonic ventricular zone (Figure 5 and data not shown). Addition of SDF1α significantly increased cell division in primary dissociated cultures of the wild-type e13.5 cerebellar anlage. Blocking SDF1α function using the Cxcr4 receptor antagonist AMD 3100 (Rosenkilde et al., 2004), significantly inhibited BrdU uptake in these cultures (Figure 4B). The majority of cerebellar progenitors at e13.5 derive from the cerebellar ventricular zone, although both cerebellar rhombic lip progenitors and a small number of granule cell progenitors are also present at this time. To determine if the anti-proliferative AMD3100 could specifically alter cerebellar ventricular zone proliferation, we cultured whole e13.5 cerebellar anlage explants in serum containing media with or without AMD 3100 for 24 hr. Subsequent sections of these whole mount cerebellar cultures revealed that blockage of the SDF1α-Cxcr4 signalling pathway caused a significant increase in ventricular zone neuronal differentiation and reduced proliferation. (Figure 4A,C–F).10.7554/eLife.03962.008Figure 4.SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.


Foxc1 dependent mesenchymal signalling drives embryonic cerebellar growth.

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

SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.(A) Schematic of dorsal whole mount view of the embryonic mouse head and a brief description of experiments included in the figure. (B) Graph showing the percentage of BrdU+ cells in primary dissociated cerebellar culture. Addition of SDF1α significantly increased BrdU uptake by cells. Addition of AMD3100, an antagonist of SDF1α significantly reduced BrdU incorporation. (C, D; C′, D′) Sagittal sections of e13.5 cerebellum cultured whole mount for 1 day and stained for Ki67 (inset) and β-III Tubulin. Addition of AMD3100 to the culture significantly reduced cell proliferation and increased differentiation in the ventricular zone (D). (E) Graph showing the percentage of Ki67 and (F) β-III Tubulin+ cells in the cerebellar ventricular zone. (G) Schematic describing the transwell migration assay. Addition of SDF1α significantly increased the number of cells migrating through the membrane of the insert. Addition of AMD3100 to the upper well significantly lowered the number of migrating cells (H). (H) Graph quantifying the results of the experiment described in (G) (I) Schematic of a sagittal section of the e13.5 cerebellum. Ventricular zone progenitors are labelled in yellow, while Ptf1a positive cells that exit the ventricular zone are marked red. RL progenitors are labelled light green while granule cell progenitors exiting the RL are labelled dark green. (J–K′′) Matrigel assay to study the effect of SDF1α on the neuronal migration. Ptf1a positive cells from an e13.5 cerebellar slice, when incubated with SDF1α coated acrylic beads were seen to move towards the source of the chemokine (K–K′′). Saline coated beads had no effect on migration (J, J′). Abbreviations used; VZ—Ventricular Zone, RL—Rhombic Lip, EGL—External Granular Layer and Mes—Mesenchyme. The white dotted line in (C–D′) represents the cerebellar ventricular surface. Scale bar = 100 µm. In Graphs (B), (E, F) and (H), data is represented as mean percentage of BrdU or β-III Tubulin positive cells or migrating cells ± s.e.m. In (B) and (H) ** indicates significance with respect to Control, while *** indicates significance with respect to SDF1α treatment. In (E, F) *** indicates significance with respect to Control. (p < 0.05), for all data.DOI:http://dx.doi.org/10.7554/eLife.03962.008
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fig4: SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.(A) Schematic of dorsal whole mount view of the embryonic mouse head and a brief description of experiments included in the figure. (B) Graph showing the percentage of BrdU+ cells in primary dissociated cerebellar culture. Addition of SDF1α significantly increased BrdU uptake by cells. Addition of AMD3100, an antagonist of SDF1α significantly reduced BrdU incorporation. (C, D; C′, D′) Sagittal sections of e13.5 cerebellum cultured whole mount for 1 day and stained for Ki67 (inset) and β-III Tubulin. Addition of AMD3100 to the culture significantly reduced cell proliferation and increased differentiation in the ventricular zone (D). (E) Graph showing the percentage of Ki67 and (F) β-III Tubulin+ cells in the cerebellar ventricular zone. (G) Schematic describing the transwell migration assay. Addition of SDF1α significantly increased the number of cells migrating through the membrane of the insert. Addition of AMD3100 to the upper well significantly lowered the number of migrating cells (H). (H) Graph quantifying the results of the experiment described in (G) (I) Schematic of a sagittal section of the e13.5 cerebellum. Ventricular zone progenitors are labelled in yellow, while Ptf1a positive cells that exit the ventricular zone are marked red. RL progenitors are labelled light green while granule cell progenitors exiting the RL are labelled dark green. (J–K′′) Matrigel assay to study the effect of SDF1α on the neuronal migration. Ptf1a positive cells from an e13.5 cerebellar slice, when incubated with SDF1α coated acrylic beads were seen to move towards the source of the chemokine (K–K′′). Saline coated beads had no effect on migration (J, J′). Abbreviations used; VZ—Ventricular Zone, RL—Rhombic Lip, EGL—External Granular Layer and Mes—Mesenchyme. The white dotted line in (C–D′) represents the cerebellar ventricular surface. Scale bar = 100 µm. In Graphs (B), (E, F) and (H), data is represented as mean percentage of BrdU or β-III Tubulin positive cells or migrating cells ± s.e.m. In (B) and (H) ** indicates significance with respect to Control, while *** indicates significance with respect to SDF1α treatment. In (E, F) *** indicates significance with respect to Control. (p < 0.05), for all data.DOI:http://dx.doi.org/10.7554/eLife.03962.008
Mentions: We hypothesized that Foxc1 regulates the expression of secreted factors in the head mesenchyme which then non-autonomously influence the developing cerebellum. Based on candidate gene analysis, we previously reported that Foxc1−/− mutant posterior fossa mesenchyme has reduced expression of SDF1α (Cxcl12), Bmp2 and 4 (Aldinger et al., 2009). We tested the ability of these secreted factors to influence cerebellar ventricular zone proliferation and migration of ventricular zone-derived neurons using various assays (Figure 4). The embryonic cerebellar ventricular zone does not express Bmp-receptors although they are expressed in the rhombic lip (Machold et al., 2007). In contrast, the SDF1α receptor, Cxcr4 is highly expressed in the embryonic ventricular zone (Figure 5 and data not shown). Addition of SDF1α significantly increased cell division in primary dissociated cultures of the wild-type e13.5 cerebellar anlage. Blocking SDF1α function using the Cxcr4 receptor antagonist AMD 3100 (Rosenkilde et al., 2004), significantly inhibited BrdU uptake in these cultures (Figure 4B). The majority of cerebellar progenitors at e13.5 derive from the cerebellar ventricular zone, although both cerebellar rhombic lip progenitors and a small number of granule cell progenitors are also present at this time. To determine if the anti-proliferative AMD3100 could specifically alter cerebellar ventricular zone proliferation, we cultured whole e13.5 cerebellar anlage explants in serum containing media with or without AMD 3100 for 24 hr. Subsequent sections of these whole mount cerebellar cultures revealed that blockage of the SDF1α-Cxcr4 signalling pathway caused a significant increase in ventricular zone neuronal differentiation and reduced proliferation. (Figure 4A,C–F).10.7554/eLife.03962.008Figure 4.SDF1α induces cell division in cerebellar ventricular zone progenitors and also functions as a chemoattractant.

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.

Show MeSH
Related in: MedlinePlus