Gcps of two altered anchoring centers in mutants exhibit the same coordinated changes as in WT

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> Phalloidin staining (red) shows gcp morphology in sagittal sections of P0 WT (a, e) and mutant mice (c, g). At P0, in the WT secondary fissure (a), gcps are more elongated (ci = 0.55) than gcps in the area of the prepyramidal fissure (e) (ci = 0.71). In contrast, in mutants, the gcps in the secondary (c) and prepyramidal (g) fissures are similarly elongated. Dotted white line outlines the EGL, based on the anti-Pax6 immunostaining of adjacent sections to depict the thickness of EGL. Asterisks indicate the base of the fissure. Ci of gcps (gray is WT, white is mutant). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences between the base of the fissure and crown of the folia for each data set (< 0.0009 for P0; < 0.0002 for P1). Anti-pH3 immunostaining at P0 reveals that there are more gcps in mitosis in the areas where the prepyramidal and secondary fissures will form than at the crown of folium VIII. Bar graphs depict quantification of the number of pH3 positive gcps in the prepyramidal fissure (light blue bars) and in the secondary fissure (dark blue bars) compared to the crown of the intervening folium (red bars). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences between the two regions (< 0.004 for P0 for the prepyramidal fissure; < 0.05 for P0 for the secondary fissure). Scale bar: 15 μm

Purkinje cell layer folding indicates the future positions of the base of each principal fissure

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> () At E16.5 the mouse Cb has a smooth surface, but anti-Calbindin immunostaining (red) shows a multilayer of Pcs with invaginations in the areas where fissures will form (asterisks). Yellow asterisk indicates the fissures shown in (d, e, f). () At E17.5 (b, e) and E18.5 (c, f) both the Pc layer and outer surface invaginate (foliate) simultaneously. Anti-Pax6 immunostaining shows accumulation of the gcps in the EGL, above the Pc layer invagination at E16.5 and E17.5 (inset in (d, e)), whereas by E18.5 the EGL is similar in thickness at the base and at the crown of the folia (inset in (f)). () In animals, is expressed ubiquitously in precursors. Upon administration of tamoxifen at E12.5, ER translocates to the nucleus, where it recombines the floxed STOP signal in the locus. Sagittal sections of E17.5 Cb show that some fate mapped cells (green) colocalize with anti-RORα (red) and are, therefore, Pcs. Marked Pcs (white arrows) have round cell bodies and have extended their axons in various directions. Scale bars: (a-c) 100 μm; (d-f) 40 μm; (g-g2) 15 μm

Cerebellar foliation is executed by anchoring the base of the fissures and by folia lengthening

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> The Cb vermis primordium at E16.5 is a smooth structure (a), which, at E17.5 (b), is divided into four lobes by three fissures (asterisks). At E18.5 the four principal fissures (asterisks) and five cardinal lobes (dotted outlines) are evident. The cardinal lobes are designated as anterobasal (red outline), anterodorsal (green outline), central (blue outline), posterior (yellow outline) and inferior (white outline). The adult Cb has ten lobules that develop from the cardinal lobes, marked by colored outlines in (c, d) (I-X, principal fissures are marked by asterisks). Superimposition of midline vermis tracings from E16.5 to P21 show that the folia grow by lengthening, while the bases of the fissures are largely fixed in position. Mb, midbrain; Cb, cerebellum; pc, preculminate; pr, primary; sec, secondary; pl, posterolateral fissure. Scale bars: (a-c) 300 μm; (d) 500 μm; (e) 600 μm

Gcps in the emerging fissures have a shorter mitotic index than other gcps

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> Medial sagittal sections of E17.5 (a) and E18.5 (b) embryos treated for approximately 20 minutes with BrdU. Anti-BrdU (green) immunostaining shows uniform BrdU incorporation throughout the EGL (asterisks depict locations where fissures will form). Quantification of the number of pH3 positive gcps in the region where the EGL is thicker (blue bars) versus the intervening thinner regions (red bars). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences between the two regions (< 0.001 for E16.5, < 0.0001 for E17.5). Anti-pH3 immunostaining at E16.5 (data not shown) and E17.5 indicates that there are more gcps in mitosis in the regions where future fissures will form (areas between dotted outline) than in other regions. By E18.5 pH3-positive cells are distributed equally throughout the EGL (between dotted lines). Anti-Calbindin was used to mark the Pc layer. Arrows indicate pH3 positive cells in the EGL. Scale bars: (a, b) 100 μm; (d, e) 50 μm

Correct timing of anchoring center initiation is required for correct folial shape

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> Superimposition of midline vermis tracings at E17.5, P0, P3 and adult show that the delay in anchoring center formation of the secondary fissure and premature anchoring center formation of the prepyramidal fissure in mutants result in the a misshapen lobule VIII. Grey arrows demarcate prepyramidal fissure; black arrows demarcate secondary fissure

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.This work was supported by grants from the Wellcome Trust (080470) to M.A.B. and (072111) to M.A.B. and I.M., by the Medical Research Council and a Leverhulme Trust Fellowship to I.M., by the EU research program (LSHG-CT-2004-512003 and MEIF-CT-2006-025154), the Spanish Science Program (MEC BFU2005-09085, RD06/0011/0012), the ELA Foundation Research and TV3 LA (MARATO-062232) to D.E. and S.M., and by the National Institutes of Health (R01 HD050767) to A.L.J. and (R01 CA78711) to G.R.M.Peer Reviewe