Figure 1.
The strategy for administering caffeine to early chick embryo in vivo (A) and normal chick embryo neurulation (B–D).
A: Schematic drawing of caffeine introduction into early chick embryo in vivo. The fertilized egg was windowed on day 1.5, treated with caffeine, then sealed and continually incubated until the required stage. B–D: Photographed images of a normal chick embryo development, taken at the incubation 25, 30 and 35 hours. The movie version can be found in the Movie S1. The white arrows indicate the process of neural tube closure. Scale bar = 500 µm in B–D. Abbreviation: Nt, neural tube.
Figure 2.
Failure of neural tube closure in the prosencephalon after administering caffeine to chick embryo.
A: The control embryo was treated with physiological saline solution, and then photographed as a whole-mount embryo and transverse sections at the head and trunk levels, which is indicated by the dotted line (A1 and A2). The black arrowheads indicate the point of neural tube closure, hence neural tube formation. The green arrowheads indicate the development of somites. B–D: 0.5, 1 and 1.5 mg/ml caffeine-treated embryos photographed as whole mount embryos. Transverse sections were taken at the head ad trunk levels as indicated by the dotted lines (B1–D2). The transverse sections show the failure of neural tube closure and thickening of the head mesenchymal layer in the prosencephalon (B1–D1), which are indicated by the black arrowheads. Also, the cells of the somites appear more scattered in comparison to the control, as indicated by the green arrows (B2–D2). The process of neurulation is completely inapparent in the high caffeine concentration groups, as indicated by the flatness of the neural plates (C–D). The black horizontal lines on neural tubes (A2–D2) indicate the width of neural tubes. Scale bar = 1 mm in A–D and 100 µm in A1–D2. Abbreviation: Nt, neural tube.
Figure 3.
Caffeine induced discontinuity in cranial neural crest cells migration during delamination.
A: The control embryo, which was treated with physiological saline solution. B–D: Caffeine-treated embryos of different concentrations. A1–D1: Transverse sections of the control and caffeine-treated embryos at the cranial level. The morphology of the embryos appears normal at caffeine concentrations of under 1.5 mg/ml, above which the structure of the neural tube is damaged (D1). A2–D2: Transverse sections of whole-mount immunocytochemistry against HNK1. In the caffeine-treated embryos, the HNK1 labeled migratory neural crest cells showed a disjointed migration trajectory, as indicated by the blue dotted arrows (B2–D2). The normal continuous migration trajectory was demonstrated by the control (A2). A3–D3: Transverse sections of whole-mount immunocytochemistry for Pax7. In the caffeine-treated embryos, the Pax7 labeled neural crest cells followed a relatively normal migration trajectory (B3–C3). However, at 1.5 mg/ml concentration of caffeine, there is some structural damage as indicated by the white arrows in D3. A4–D4: Images of HNK1 (A2–D2) merged with Pax7 (A3–D3). Scale bar = 100 µm in A1–D4. Abbreviation: Nt, neural tube.
Figure 4.
Caffeine induced discontinuity in trunk neural crest cells migration during delamination.
A: The control embryo, which was treated with physiological saline solution. B–D: Caffeine-treated embryos of different concentrations. A1–D1: Transverse sections of the control and caffeine-treated embryos at the trunk level. The morphology of the embryos appears normal at caffeine concentrations of under 1.5 mg/ml, above which the structure of the neural tube is damaged (D1). A2–D2: A2–D2: Transverse sections of whole-mount immunocytochemistry against HNK1. In the caffeine-treated embryos, the HNK1 labeled migratory neural crest cells showed a disjointed migration trajectory, as indicated by the blue dotted arrows (C2–D2). The normal continuous migration trajectory was demonstrated by the control (A2). A3–D3: Transverse sections of whole-mount immunocytochemistry for Pax7. In the caffeine-treated embryos, the Pax7 labeled neural crest cells followed a relatively normal migration trajectory (B3–C3). However, at 1.5 mg/ml concentration of caffeine, there is some structural damage as indicated by the white arrows in D3. A4–D4: Images of HNK1 (A2–D2) merged with Pax7 (A3–D3). Scale bar = 100 µm in A1–D4. Abbreviation: Nt, neural tube.
Figure 5.
Caffeine induced suppression of neuron proliferation and differentiation.
A–D: Neurofilament expression pattern of normal developing neural tubes in the whole-mount embryo (A) and transverse sections (B-bright-field; C-fluorescence; D-merge). The white arrowheads indicate neurofilament positive neurons in the neural tube. E–H: The disassociated chick embryo brain cells were treated with different concentrations of caffeine for 24 hours. Following which, the number of neurofilament positive cells in the caffeine-treated was reduced in comparison to the control (E,I). Also, the neuron length decreased after caffeine treatment (J). Scale bar = 100 µm in A, 50 µm in B–D and 100 µm in E–H.