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Development
2014 Jul 01;14114:2791-5. doi: 10.1242/dev.101758.
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Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression.
Butts T
,
Hanzel M
,
Wingate RJ
.
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The cerebellum has evolved elaborate foliation in the amniote lineage as a consequence of extensive Atoh1-mediated transit amplification in an external germinal layer (EGL) comprising granule cell precursors. To explore the evolutionary origin of this layer, we have examined the molecular geography of cerebellar development throughout the life cycle of Xenopus laevis. At metamorphic stages Xenopus displays a superficial granule cell layer that is not proliferative and expresses both Atoh1 and NeuroD1, a marker of postmitotic cerebellar granule cells. Premature misexpression of NeuroD1 in chick partially recapitulates the amphibian condition by suppressing transit amplification. However, unlike in the amphibian, granule cells fail to enter the EGL. Furthermore, misexpression of NeuroD1 once the EGL is established both triggers radial migration and downregulates Atoh1. These results show that the evolution of transit amplification in the EGL required adaptation of NeuroD1, both in the timing of its expression and in its regulatory function, with respect to Atoh1.
Fig. 1. Xenopus displays a non-proliferative EGL at metamorphosis. Schematic drawings of tadpole (stage 48: A-H) and froglet (stage 58: I-O) stages of development are shown with corresponding brain profiles [cerebellum (cb) in red] and location of whole-mount and section views (blue boxes). (A) Atoh1 expression in whole-mount hindbrain (hb) and cerebellum. (B) Mitotic cells in the rhombic lip (rl; blue dotted line) in an equivalent embryo stained for PH3. (C) Cerebellum of embryo in A (boxed region) counterstained for PH3. The anterior extra-cerebellar Atoh1-postive regions (asterisk, also in D,H) correspond to the primordium of isthmic nuclei. (D) In sagittal section, PCNA staining shows that the cerebellum anlage (white line) is devoid of superficial proliferative neurons. (E) Barhl1 is expressed in cerebellum (arrow) and hindbrain. (F) Lhx9 is expressed in hindbrain only. (G) NeuroD1 is expressed in cerebellum (arrow). (H) In sagittal section, Atoh1 is not expressed on the surface of the cerebellum anlage (black line). (I) In the froglet, mitotic cells in the cerebellum are still confined to the rhombic lip. (J) Zic1, a marker of granule neurons at all stages of development, is expressed in both an internal granule cell layer (igl) and an external germinal layer (egl, arrow). (K) Atoh1 is also expressed in the EGL. (L) NeuroD1 is expressed in both layers. (M) PCNA staining in sagittal section confirms that the EGL is non-proliferative. (N) Calbindin is expressed in the Purkinje cell layer (pcl). (O) Purkinje cells do not express Shh. mb, midbrain; fb, forebrain.
Fig. 2. NeuroD1 expression at E4 abrogates proliferation and alters migration paths of granule cell precursors in chick. GFP:IRES:NeuroD1 (or GFP-only control) was electroporated into the chick cerebellar rhombic lip at E4 and the cerebellum analysed at E6-8. (A) Surface view of the EGL in a whole-mount E8 cerebellum anlagen expressing GFP:IRES:NeuroD1 and stained for PH3 (red). (B) GFP signal (green). (C) Merged PH3 and GFP images. (D) PH3 (red)-labelled section through a control GFP-electroporated cerebellum at E8. Yellow cells are proliferating granule precursors in the EGL. (E) GFP:IRES:NeuroD1 expression drives cells from the EGL and none is co-stained for PH3. (F) Timecourse of migration of GFP:IRES:NeuroD1-expressing cells from the rhombic lip at E6, E7 and E8 in sagittal section counterstained for PH3 (red).
Fig. 3. NeuroD1 regulation is conserved across tetrapods, but its activity is modified in the amniote lineage. Confocal microscopy images of sagittal sections of E8 chick cerebellum stained for GFP (green) and PH3 (red) following electroporation with: (A) a control plasmid encoding GFP, (B) the CNE upstream of the mouse basal NeuroD1 promoter and (C) the CNE upstream of the Xenopus basal NeuroD1 promoter. (D) Percentage of GFP-labelled cells in the EGL following electroporation of control (n=15), mouse (n=20) and Xenopus (n=12) CNE constructs. The difference between the control and either the mouse or Xenopus construct is highly significant (P<0.0001). Expression of the basal promoter of mouse alone is unable to drive the expression of GFP (data not shown). (E) Atoh1 reporter driving the expression of mCherry (red), co-expressed at E14 with NeuroD1 (mouse CNE) GFP reporter (green) labels granule cells in the EGL. After 24 h in culture, a large proportion of cells expressing NeuroD1 retain perdurant mCherry (red), indicating that they had previously activated Atoh1. Most have yet to leave the EGL. (F) By contrast, when NeuroD1 is co-expressed with the Atoh1 reporter, no mCherry (red) can be detected and the majority of cells have exited the EGL, indicating that NeuroD1 suppresses the activity of the Atoh1 enhancer.
