First published online October 28, 2005
doi: 10.1242/10.1242/dev.02075
Development 132, 5069-5079 (2005)
Published by The Company of Biologists 2005
Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina
Leanne Godinho1,
Jeff S. Mumm1,
Philip R. Williams1,
Eric H. Schroeter1,
Amy Koerber1,
Seung W. Park2,
Steven D. Leach2 and
Rachel O. L. Wong1,*
1 Department of Anatomy and Neurobiology, Washington University School of
Medicine, 660 South Euclid Avenue, Box 8108, St Louis, MO 63110, USA
2 Departments of Surgery, Oncology and Cell Biology, Johns Hopkins Medical
Institutions, Baltimore, MD 21287, USA
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Fig. 1. ptf1a regulatory elements drive expression of GFP in amacrine
cells in transgenic zebrafish. (A) Confocal images of a cryosectioned retina
from a ptf1a::GFP fish at 76 hpf reveal a population of
GFP+ cells in the inner portion of the INL and a population of
cells in the GCL that are immunoreactive for the 5E11 antigen, suggesting they
are amacrine cells (arrows in the GCL indicate displaced amacrine cells). The
arbors of both amacrine cell populations ramify in the IPL, forming a
laminated plexus. Horizontal cells (H) located in the outer part of the INL
also express GFP. (B) In vivo confocal time-lapse images of a vitreally
located GFP+ amacrine cell co-labeled with membrane-targeted
mCherry (red) driven by an  -tubulin promoter reveal that its neurites
are oriented towards the INL. (C) In vivo confocal time-lapse images reveal
that both populations of GFP+ amacrine cells (green) can be
identified as early as 39 hpf. The vitreally located GFP+ cells
with rounded or flattened somata (arrows) become displaced to the GCL as a
plexus forms (black arrowheads, 51 hpf) between them and the INL population of
GFP+ amacrine cells (46 and 51 hpf). Counterstaining with BODIPY
Texas Red demonstrates that the GFP+ plexus lies within the forming
IPL. Gray-scale images of GFP+ amacrine cells are shown to permit
better visualization of GFP+ processes.
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Fig. 2. The pax6-DF4 enhancer element drives expression of fluorescent
proteins in transgenic lines of zebrafish. In most of the lines generated,
expression of membrane-targeted fluorescent proteins was confined to the
retina (A-D). Confocal images of larval zebrafish from CFP line Q11 (A) and
YFP line Q14 (C) reveal that at early stages, expression within the retina is
in clones of cells that span the neuroepithelium. With time, expression
becomes confined to amacrine cells located in the INL with processes ramifying
in the IPL. The number of labeled amacrine cells varied, but was sometimes
sparse enough to allow individual amacrine cells to be visualized
unambiguously (B,D). Ubiquitous expression throughout the embryo was observed
in CFP line Q01 (E). In the retina of line Q01, cells in the nuclear layers
were outlined and the plexiform layers appeared to be densely labeled (F).
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Fig. 3. Migrating amacrine cells show undirected neurite outgrowth. Time-lapse
images of a YFP+ amacrine cell (arrow) in line Q14 migrating
towards the GCL. Multiple neurites emerge from the amacrine cell, but they do
not appear to be polarized towards the GCL. Imaging commenced at 51 hpf
(0'). The dashed line represents the position of the future IPL, at the
interface between the forming INL and GCL. Clones of FP-expressing cells (like
those to the right of the migrating amacrine cell) are usually seen in the
pax6 transgenic lines at early stages of development.
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Fig. 4. Exuberant neurite outgrowth continues even when amacrine cells are located
near or at their final somal positions. (A) Time-lapse confocal images of a
pair of CFP+ amacrine cells from line Q11 reveal neurites directed
appropriately towards the GCL, and erroneously towards the outer retina.
Imaging commenced at 56 hpf (0') and proceeded to 64 hpf (480').
To examine whether the neurite extension of amacrine cells located near their
final somal positions displayed directionality, we scored neurites as being
directed towards the GCL, the OLM or sideways (S), as shown for the amacrine
cell in B. For the cell in B, during the period of recording, the number of
neurite tips directed towards the GCL was consistently found to exceed the
number of neurite tips directed elsewhere (C). (D) Histograms of a similar
analysis for six cells (each differently shaded), where the number of neurites
was averaged over a period of 90 minutes.
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Fig. 5. Amacrine neurites directed towards the GCL or the OLM show similar motility
rates but significantly different life times. (A) Changes in neurite length
for an OLM-directed process (1) and GCL-directed processes (2,3) of an
amacrine cell (CFP+ line Q11, 54 hpf) over a period of 30 minutes
are plotted in B. Such measurements were used to calculate average motility
rates (C). Average extension rates for processes directed towards the OLM were
found to be 0.17±0.05 µm/minute, and toward the GCL 0.19±0.06
µm/minute. Average retraction rates from the OLM were found to be
0.18±0.05 µm/minute and from the GCL 0.18±0.04 µm/minute.
These rates were not significantly different (n=9 cells; 8-19
processes per group; P>0.05 for all groups, Mann-Whitney rank sum
test). (D) A significant difference was found between the life times of
processes directed towards the GCL (n=17 processes) and the OLM
(n=15 processes; P<0.001, Mann-Whitney rank sum
test).
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Fig. 6. Analysis of the dynamic behavior of amacrine neurites within the IPL
reveals extensive remodeling. Changes in neurite length over a period of 30
minutes were measured for amacrine cells with extensive lateral arbors in the
IPL and used to calculate average motility rates. (A) Changes in length for
three neurites (1,2,3) from the lateral arbor of an amacrine cell
(CFP+ line Q11, 58 hpf) are plotted in B. (C) Average extension and
retraction rates of processes within the IPL were found to be similar
(0.16±0.06 µm/minute and 0.15±0.04 µm/minute,
respectively; n=3 cells, 24 processes; P>0.05;
Mann-Whitney rank sum test). (D) Time-lapse images of an amacrine cell (54 hpf
at 0') digitally rotated to provide a clearer view of its lateral arbor.
Extensions (e) and retractions (r) of neurites within the IPL resulted in
dramatic changes of the territory occupied by the lateral arbor (see E).
Occasionally, neurite outgrowth towards the outer retina, from the lateral
arbor (o at 0') or from the cell body (o at 80') was observed. (E)
Time-lapse images of the amacrine cell in D digitally rotated to provide en
face views of the lateral arbor. The extent of the cell's lateral territory is
outlined in white and the location of the presumed Golgi (bright spot in the
cell body in D) is depicted by a black oval.
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Fig. 7. Amacrine cells that ultimately stratify in the OFF sublamina display an
early bias for the outer half of the IPL. (A) Confocal time-lapse images of a
YFP+ amacrine cell from line Q08 (green) in the background of the
ubiquitously-expressing CFP line Q01 (red). The amacrine cell confines its
lateral arbors to the outer part of the IPL from the earliest time points (57
hpf) until the time when it is stratified (80 hpf). (B) Early restriction of
neurites to the outer half of the IPL for OFF amacrine cells can be better
appreciated for a cell (GFP line 244, green) that arrives at the interface of
the IPL (right cell in all panels) demarcated by the ubiquitous expression of
CFP (line Q01, red). (C) Morphometric analysis of 10 GFP+ or
YFP+ cells imaged in the background of line Q01 confirmed that the
vast majority of GFP+ or YFP+ pixels lies in the outer
half of the IPL early (57-62 hpf) and late (70-72 hpf), for presumed OFF
amacrine cells.
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Fig. 8. Amacrine cells display an early bias for the IPL sublamina in which they
will ultimately stratify. (A) Confocal time-lapse images of a pair of
GFP+ amacrine cells from line 220 (green) in the background of the
ubiquitously expressing CFP line Q01 (red). The cell that eventually
stratifies in the ON sublamina of the IPL (cell to the right) ramifies its
neurites in the inner half from the outset. Similarly, the cell that
eventually stratifies in the OFF sublamina (cell to the left) restricts its
neurites to the outer half of the IPL from the outset. (B) Time-lapse of a
CFP+ amacrine cell (line Q11, red) in the background of GFP line
220 (green) in which populations of OFF and ON amacrine cells are labeled.
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© The Company of Biologists Ltd 2005
