Figure 7

ECRG4 gene knockdown causes enlarged hindbrain ventricles and a hyper proliferative response in the developing zebrafish. A: Development of Zebrafish after morpholino injection 48 hours post fertilization. In control (A1) and Ecrg4 (A2) morpholino treatments, the head, eyes, heart, yolk and hind ventricle (arrows) were photographed for analysis. The anti-sense Ecrg4 a morpholino caused readily detectable hindbrain ventriculomegaly resembling a hydrocephalus-edema-like response in the CNS. The ventricles can be isolated from images for analyses (n = 8). B: Quantification of developmental effects of morpholino injection. Hindbrain ventricle size was quantified by pixel area of microscope images from n = 8 control (MO ctl), n = 8 Ecrg4 a MO-treated embryos or (n = 8) Ecrg4 a MO co-injected with the Zebrafish mRNA ortholog A to neutralize specific inhibition (error bars: mean ± standard deviation). C-E: Cell growth in Zebrafish 24 hours after control or Ecrg4a morpholino injection 24 hours post fertilization. Proliferating cells in control MO (top panels) from sagittal (C1), dorsal (D1) or after cross sections at dashed line (E1) can be compared to similar sections of Ecrg4a MO (bottom panel) injected animals by staining for phosphorylated Histone 3 (H3P, green), a marker of cell growth. H3P-positive proliferating cells are compared to that of cytoplasmic glial fibrillary acidic protein (red). The arrow (D2) highlights space corresponding to enlarged ventricles in MO-Ecrg4 treated embryos. The H3P positive cells are on the ventricle surface and there are more H3P cells and thicker GFAP staining in Ecrg4a MO. Scale bar is 100 μm F Quantification of cell growth in Zebrafish after control or Ecrg4a morpholino injection. The amount of H3P-postive cell staining was determined as described in the text and compared between treatment groups (n = 7, mean ± SEM). There were significantly more labeled cells in the Ecrg4a morpholino group. Scale bar is 100 μm.