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doi: 10.1242/10.1242/dev.00569

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Monitoring neural progenitor fate through multiple rounds of division in an intact vertebrate brain

Centre for Cellular and Molecular Dynamics, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (83K): [in a new window]   Fig. 1. Dynamics of hindbrain neurogenesis. (A-D) Transverse sections through the hindbrain at the level of rhombomeres 4 and 5 in HuC-GFP-positive embryos. The GFP fills both neuronal cell bodies and processes. Broken lines in B-D indicate the boundary between the mantle zone and the marginal zone. A distinct T-shaped ventricular zone is obvious at 36 hpf as an area devoid of HuC-GFP expression and this decreases dramatically by 48 hpf. Dorsal is to the top. (E) Horizontal confocal section through a living HuC-GFP-positive embryo stained with Texas Red Bodipy Ceramide to outline cell profiles. Arrowheads indicate the boundary between rhombomeres 3 and 4 and arrows indicate the boundary between rhombomeres 5 and 6. The embryonic midline is highlighted by a broken line. (F) Graph documenting numbers of neurons and non-neurons in rhombomeres 4 and 5 throughout embryogenesis. The red curve highlights the increase in neurons and the blue curve indicates the number of non-neuronal cells. Scale bars: 50 µm.

View larger version (92K): [in a new window]   Fig. 2. Observations of two clones expanding throughout embryogenesis. (A) Dorsal view of an embryo at 20 hpf. Two elongated progenitor cells are seen, one on each side of the embryonic midline. Scale bar: 20 µm. (B) Dorsal view of the same specimen at 28 hpf. Both cells have divided. On the left-hand side daughter cells remain close to the ventricular zone, whereas on the other side two cells are seen further from the ventricular surface, suggesting they will become neurons. Scale bar: 20 µm. (C) Lateral view of cells on the left-hand side of the midline at 38 hpf. Four cells are now visible, suggesting that both cells seen at 28 hpf divided once more. The ventricular surface is indicated by a broken line. Scale bar: 20 µm. (D) Lateral view of cells on the right-hand side of the midline at 38 hpf. These two cells have moved further from the ventricular zone, indicating a neuronal phenotype. The ventricular surface is indicated by a broken line. Scale bar: 20 µm. (E) Confocal projection of the specimen fixed at 48 hpf. There are four cells (see inset) on the left side of the embryonic midline and two on the right, showing that none of the cells observed at 38 hpf divided. Transverse section with dorsal to the top. Scale bar: 50 µm. Arrows in inset indicate two cell bodies very close together. (F) Same section as in E but revealing underlying HuC-GFP expression. All dextran-labelled cells express HuC-GFP, indicating they are neurons. The ventricular surface is indicated by a broken line. Scale bar: 50 µm. (G) Magnified image of four cells on the left side of the midline showing all four co-express HuC-GFP and dextran. (H) Same image as in G showing the GFP channel alone.

View larger version (76K): [in a new window]   Fig. 3. Neurons can be phenotyped by morphology and/or HuC-GFP expression. (A,B) Dorsal view of four neurons at 36 hpf in the live embryo. Scale bars: 20 µm. (A) Examining the cells by means of dextran visualization alone does not provide conclusive evidence of cell phenotype. (B) Coincidence of dextran-labelled cells with HuC-GFP confirms the cells' phenotype as neuronal. (C) Dorsal view of four neurons at 36 hpf in another live embryo. These four cells can be phenotyped by morphology alone by virtue of their axons. Scale bar: 20 µm. (D) Confocal projection of a clone in a fixed embryo at 48 hpf. Four cells with a neuronal morphology are seen on the right-hand side of the embryonic midline and two cells with a neuronal morphology on the left-hand side. Transverse view with dorsal to the top. Scale bar: 50 µm. (E) Confocal projection of the same specimen as documented in D shows coincident expression of all labelled cells with HuC-GFP. Here we phenotyped cells by morphology and transgene expression. Scale bar: 50 µm.

View larger version (48K): [in a new window]   Fig. 4. Direct observation of an asymmetric division. (A) Single cell at 24 hpf labelled with dextran is shown in red. The cell has retracted its process from the pial surface, indicating that it is about to divide at the ventricular surface. Three non-related neurons expressing HuC-GFP are seen at the pial surface. Arrowhead points to ventricular surface. Scale bar: 10 µm. (B-D) Lateral views of the same specimen as in A. Anterior is to the right and dorsal to the top. Arrowheads point to ventricular surface throughout. All scale bars: 20 µm. (B) Two cells are present at 36 hpf following the earlier division. One has a neuronal morphology and one a radial morphology, which may indicate that it is still in the cell cycle. (C) Three neurons with axonal processes are present at 48 hpf, showing that the radial progenitor cell divided again. (D) Same view as C but with brightfield overlay.

View larger version (9K): [in a new window]   Fig. 5. Summary of lineage trees terminating in neurons only. (A) The composition and frequency of lineage trees observed until 48 hours that produced neurons only. Neurons generated by an asymmetric division are indicated in italics and by an asterisk. (B) Position of 66 neurons taken from clones analysed at 48 hpf projected onto a schematic transverse section through the hindbrain. Neurons are spread over most areas of the mantle layer.

View larger version (70K): [in a new window]   Fig. 6. Orientation of division is almost always within the plane of the ventricular zone. (A-C) Sequence from a time-lapse movie showing a dextran-labelled cell divide along the plane of the ventricular zone. Dorsal view with anterior to the top. Scale bars: 20 µm. (D-E) Sequence from a time-lapse movie illustrating a cell rounding up in the ventricular zone and undergoing mitosis (arrowheads). In this case cells are stained with the vital dye Bodipy 505/515. Dorsal view with anterior to the top. Scale bars: 20 µm.

View larger version (59K): [in a new window]   Fig. 7. Non-neuronal cells in clones at 48 hpf. (A) Transverse section through a clone in a HuC-GFP-positive embryo. One cell can be seen near the ventricular surface (arrowhead) outside of the HuC-GFP-positive area. Dorsal is to the top. Scale bar: 50 µm. (B) Lateral view of a clone with three radial cells and a neuron. Arrowhead points to the ventricular zone. Scale bar: 10 µm. (C) Typical morphology of a radial cell at 48 hpf. A distinct endfoot is seen at both the ventricular (arrowhead) and pial (arrow) surfaces. Scale bar: 10 µm. (D) Possible lineage trees for the clones containing two neurons (N) and two radial cells (R).

View larger version (88K): [in a new window]   Fig. 8. Neuronal and radial glial cells at 48 hpf. (A) Transverse section at 48 hpf labelled randomly with -tub Gal4 VP16 UAS GFP. In this specimen we see a single neuron with growth cone (arrowed) and a single cell with a radial glial-like morphology. Scale bar: 50 µm. (B) Higher magnification view of cells seen in A. Arrowheads point to radial glial processes extending to both the ventricular and pial surfaces. (C) Transverse section of hindbrain at the level of rhombomere 5. Confocal projection of neurons labelled at random with -tub Gal4 VP16 UAS GFP. Scale bar: 50 µm. (D) Confocal section through the hindbrain at 48 hpf double-stained with anti-phosphohistone H-3 (green) and anti-zrf-1 (red). Scale bar: 50 µm. (E) Higher magnification of cells expressing both phosphohistone H-3 and zrf-1. (F) Confocal section through the hindbrain at 48 hpf stained with anti-GFAP (green). Scale bar: 50 µm. (G) High magnification of a single cell expressing both phosphohistone H-3 (yellow) and anti-GFAP (green).