Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development - PubMed (original) (raw)

Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development

Xi Huang et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Cerebellar neurons are generated from two germinal neuroepithelia: the ventricular zone (VZ) and rhombic lip. Signaling mechanisms that maintain the proliferative capacity of VZ resident progenitors remain elusive. We reveal that Sonic hedgehog (Shh) signaling is active in the cerebellar VZ and essential to radial glial cell proliferation and expansion of GABAergic interneurons. We demonstrate that the cerebellum is not the source of Shh that signals to the early VZ, and suggest a transventricular path for Shh ligand delivery. In agreement, we detected the presence of Shh protein in the circulating embryonic cerebrospinal fluid. This study identifies Shh as an essential proliferative signal for the cerebellar ventricular germinal zone, underscoring the potential contribution of VZ progenitors in the pathogenesis of cerebellar diseases associated with deregulated Shh signaling, and reveals a transventricular source of Shh in regulating neural development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Shh signaling in the embryonic cerebellum. Cerebellar VZ displays Shh signaling activity by E14.5. At E16.5, Shh signaling continues to be apparent along cerebellar VZ, with higher expression in the medial portion. Note that at this stage, there are many positive cells beyond VZ within the cerebellar tissue. β-gal+ cells overlap with BLBP+ cells in E13.5 Ptch1LacZ/+ embryos.

Fig. 2.

Nestin-cre;Smof/- or Nestin-cre;SmoM2 mutants exhibit, respectively, reduced or augmented VZ progenitor proliferation and GABAergic interneuron expansion. (A) Nestin-cre activity is detected in almost all neural cells throughout the cerebellum, as indicated by whole-mount and sectioned view of X-gal staining in E13.5 Nestin-cre;R26R embryo; however, Nestin-cre;SmoM2 mutants preferentially display enhanced Shh signaling in the cerebellar VZ. (B) BrdU proliferation, CyclinD1, Pax2, and Lhx1/5 immunohistochemical analyses on WT, Nestin-cre;Smof/-, and Nestin-cre;SmoM2 mutants. Red boxes represent higher magnifications of medial and lateral cerebellar regions in adjacent panels. (C) Quantifications of marker stainings in WT, Nestin-cre;Smof/-, and Nestin-cre;SmoM2 mutants. CB, cerebellum.

Fig. 3.

Radial glia cell proliferation is defective in Nestin-cre;Smof/- and Nestin-cre;SmoM2 mutants. (A) Nestin-cre;Smof/- or Nestin-cre;SmoM2 mutants exhibit impaired or augmented BrdU incorporation in BLBP+ or Sox2+ radial glial cells, respectively. (B) Quantification of proliferating radial glial cell number in WT, Nestin-cre;Smof/-, and Nestin-cre;SmoM2 mutants. (C) Ptf1a-expressing cells are essentially not mitotic, indicated by largely nonoverlapping staining after 1-h BrdU pulse in E14.5 WT embryos. Arrow indicates one Ptf1a-expressing cell with weak, punctate BrdU signal that is rarely observed.

Fig. 4.

Shh expression is not detected in the cerebellum before E15.5. (A) Shh mRNA expression in developing cerebellum; a faint signal could be detected at E16.5 in the putative Purkinje domain beneath EGL, as indicated by arrows. (B) Indelible marking and fate-mapping of _Shh_-expressing cells in the developing cerebellum. Note lack of any GFP signal in the E13.5 Shhcre/+;mT/mG cerebella. Shhcre/+;R26R mice begin to show positive staining within the cerebellar tissue starting at E17.5. (C) Quantitative real-time RT-PCR analysis of Shh, Gli1, and Ihh expression in the developing cerebellum. Of note, Shh expression significantly differs from that of Ihh control starting at E15.5, whereas a significant level of Gli1 expression is already detected as early as E12.5.

Fig. 5.

Shh protein is present in embryonic CSF. (A) Embryonic CSF harbors Shh protein, as assayed by ELISA. The two white lines in A depict capillary tube inserted into the fourth ventricle to retrieve fresh CSF. ShhNp (starting at 0.063 nM) was used as positive control, whereas NIH 3T3 cell-conditioned media served as negative control in the analyses. (B) Primary cilia are present in proliferative radial glial VZ cells. The white frames in acetylated tubulin/Sox2 staining represent zoomed-in areas shown in the adjacent panels. Note that the white arrows indicate acetylated tubulin–labeled cilia protruding into the fourth ventricle.

Fig. 6.

Genetic ablation of Shh results in marked cerebellar VZ phenotype. (A) Wnt1-cre deleter strain drives Cre activity in hChPe, effectively ablating Shh expression selectively in hChPe but not the ventral midline of hindbrain medulla. (B) E13.5 Wnt1-cre;Shhf/- mutant cerebellar VZ shows severely impaired proliferative capacity and GABAergic and radial glia population expansion compared with WT. BrdU, Ki67, and CyclinD1 stainings indicate proliferative activity. BLBP+ and Sox2+ cells represent radial glial population. (C) Statistical comparisons between Wnt1- cre; Shhf/- mutant and WT cerebella for BrdU+ (41.5% ± 10% vs. 100% ± 12.6%, P < 0.001, n = 5), Ki67+ (37.9% ± 3.1% vs. 100% ± 6%, P < 0.001, n = 5), CyclinD1+ (35.2% ± 4.2% vs. 100% ± 9%, P < 0.001, n = 5) and Sox2+ (71.3% ± 2.9% vs. 100% ± 9.1%, P < 0.005, n = 5) cells.

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Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development - PubMed (original) (raw)