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First published online 18 February 2004
doi: 10.1242/dev.01040

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Transient requirement for ganglion cells during assembly of retinal synaptic layers

Jeremy N. Kay^1,*, Tobias Roeser^1,*, Jeff S. Mumm^2,*, Leanne Godinho^2,*, Ana Mrejeru¹, Rachel O. L. Wong² and Herwig Baier^1,

¹ Program in Neuroscience and Department of Physiology, University of California, San Francisco, 513 Parnassus Avenue Box 0444, San Francisco, CA 94143, USA
² Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA

View larger version (126K): [in a new window]   Fig. 1. Subpopulations of ACs express GFP in Pax6-DF4:mGFP transgenic zebrafish lines. (A) Schematic of inner retinal organization in mature vertebrates. Connections between ganglion cells (GCs), amacrine cells (ACs) and bipolar cells (BCs) are localized to the inner plexiform layer (IPL). The IPL is further organized into ON and OFF sublaminae that occupy approximately the inner and outer halves of the IPL, respectively. (B) The construct used to derive Pax6-DF4:mGFP transgenic lines. Expression of the Gap-43GFP fusion protein was driven by a hexamer of the 58 bp pax6 DF4 element, located downstream of the SV40 poly-A sequence, and by an upstream EF1 promoter. The sequence of the highly conserved DF4 element is shown. (C) Cross-sectional view of GFP+ ACs and their neurites in the IPL of line 220 7 dpf retina. GFP fluorescence is superimposed on a Nomarski image of a section through the eye. Two bright sublaminae are evident in the IPL (see higher magnification image, inset). (D) Digital rotation of a confocal image stack, providing an orthogonal view of a region of inner retina of a line 220 retinal wholemount at 9 dpf. S1 is an optical slice of this field of view showing sparsely distributed GFP+ neurites close to the AC cell bodies. S2-5 are image planes within the boxed region in S1. The two major GFP+ sublaminae are S2 and S4; note also sparse innervation of sublaminae S1 and S5 by GFP+ neurites. (E) Cross-sectional view of the eye of an 7 dpf line 243 fish. (F) Morphology of individual GFP+ cells in line 243. Maximum projection of a confocal image stack (47 µm total) through the inner retina of a retinal whole mount at 35 dpf. (G) Arbors of a few isolated ACs (1, 2 in F) at an IPL depth closer to the GCL. (H) Digital rotation of the complete image stack providing orthogonal views of cells 1 and 2. Cell 1 has a diffuse asymmetric arbor spanning the thickness of the IPL. By contrast, cell 2 has a radially symmetric arbor stratifying in the ON sublamina. (I-K) Cross-sections of adult line 220 retina immunolabeled for ChAT. The two bright GFP+ laminae in line 220 coincide with the two major ChAT-immunopositive bands in sublaminae S2 and S4. Some, but not all, GFP+ cells are also immunoreactive for ChAT (arrows). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NV, optic nerve; dpf, days post-fertilization; L, lens.

View larger version (140K): [in a new window]   Fig. 2. In vivo visualization of IPL formation and sublamination in Pax6-DF4:mGFP line 220. (A) GFP expression at different stages of development in line 220. At 37 hpf, expression is present in neuroblasts with processes spanning the thickness of the retina. By 54 hpf, GFP expression becomes gradually confined to ACs whose neurites take part in forming a continuous inner plexiform layer (IPL), located approximately four or five cell bodies away from the internal limiting membrane (ILM). GFP expression becomes almost exclusively confined to ACs and their neurites by 71 hpf. Note that anterior retina (left) develops before posterior. L, lens; F, choroid fissure (marks ventral retina). (B) Time-lapse confocal images showing progressive addition of GFP+ ACs over time. Each image is a z-projection of a 19 µm confocal image stack. Arrowheads indicate the same cells in all time frames. Arrow marks the approximate locations of emerging GFP-expression in ACs and neurites that contribute to the forming IPL. (C) Appearance of sublamination in the IPL. Each image is a z-projection of a 3 µm stack of confocal planes. Arrowheads indicate the same region across time points. For (B,C), dorsal retina is towards the right. Time-lapse movies of B and C (Movies 1 and 2) can be found at http://dev.biologists.org/supplemental).

View larger version (98K): [in a new window]   Fig. 3. In vivo comparison of IPL formation and sublamination in wild-type and lak mutants. Time-lapse imaging of retinas in line 220 wild-type and lakritz (lak) mutant backgrounds during IPL formation and differentiation. Both IPL formation and sublamination are delayed in the mutants. Each image is the z-projection of 3 µm confocal image stacks. Dorsal retina is towards the top. All three animals shown here were age-matched siblings, and were imaged concurrently. In all images, arrows indicate location of the developing IPL. Opposing arrows at 82 (wild type) and 85 (lak2) hpf indicate location where sublamination is apparent. Note that the IPL of lak2 becomes more organized with time. Movies showing time-lapse recordings from additional wild-type and lak siblings are provided at http://dev.biologists.org/supplemental (Movie 3A,B).

View larger version (99K): [in a new window]   Fig. 4. AC errors during IPL formation in lak mutants. (A,B) Confocal images of the same region of a lak mutant retina at 55 hpf (A) and 71 hpf (B). GFP+ AC neurites (arrows) initially accumulated near the internal limiting membrane (ILM; broken line) at various orientations, but with time, the forming IPL moved away from the ILM. Also shown in A are examples of GFP+ AC bodies (1) abutting the ILM, a feature commonly observed at this early stage in the mutants, but not apparent in wild type. (C,D) A disorganized region of mutant retina at 60 (C) and 66 (D) hpf. (C) AC neurites (arrow) are oriented abnormally, growing obliquely to the ILM. (D) An ectopic bundle of AC neurites (arrow) has formed external to the IPL. Some GFP+ neurites grow directly into this bundle rather than into the main GFP+ plexus. (E) The lak retina shown in C,D at 70 hpf, demonstrating regions of relatively normal IPL location and orientation (1); abnormal regions where there are gaps in the GFP+ plexus (2); neurites oriented perpendicular to the ILM (3); and GFP+ AC somata agglomerating in a column (4). See Movie 4 at http://dev.biologists.org/supplemental for additional views of this retina and others of similar age. (F,G) Examples of AC neurites in an 82 hpf lak retina linking neighboring cells (arrows), suggesting the tendency of ACs to grow neurites toward each other despite disorganization in their cell body locations. See Movie 5, which illustrates this tendency. All panels depict line 220.

View larger version (90K): [in a new window]   Fig. 5. Sublamination occurs in lak mutants but is locally perturbed. Confocal images of retinas of line 220 in wild-type and lak background, following IPL sublamination. At 4 and 7 days post-fertilization (dpf), two major sublaminae are observed both in wild type and in lak animals. However, sublamination is perturbed locally in the mutant. Sublaminae are some times absent (1), displaced relative to the adjacent regions (2) or added ectopically (3).

View larger version (103K): [in a new window]   Fig. 6. BC sublaminar targeting errors follow AC errors in lak mutants. Confocal images of cross sections of line 220 wild-type (wt) and lak mutant retinas (6 dpf), immunolabeled for protein kinase C (PKC). Staining is seen in BCs with axon terminals stratifying in three sublayers of the on (i.e. proximal) region of the IPL. In wild type, only one of the two major GFP+ AC sublaminae overlaps with the PKC-immunoreactive terminals, indicating that one is within the ON sublayer and the other is within the OFF sublayer. In lak, the orderly arrangement of the BC terminals is disrupted only in regions where the GFP+ AC sublaminae are perturbed. Arrows indicate a local region within which the OFF GFP+ lamina appears relatively normal (if contorted), and the ON lamina is either missing or displaced to the outer IPL. In this region, ON BC terminals (red arrowheads) are excluded from the ON sublamina, although a few puncta have aligned with diffuse GFP+ processes in the OFF sublamina (green arrowheads).

View larger version (84K): [in a new window]   Fig. 7. The lak gene is not required within ACs for dendrite morphogenesis or sublaminar targeting. (A-C) Normal morphology and monostratification of line 220 GFP+ ACs situated in wild-type retinas, as revealed by transplantation into non-GFP hosts. (A) Wild-type ACs in a wild-type host. Reconstructions of confocal stacks are shown en face and rotated 40° and 90°. Cell 1 has a symmetrical dendritic field and tightly packed processes, while Cell 2 has a larger, asymmetric dendritic field (note in the 90° rotation that the primary dendrite is already projecting to the left) and sparse, highly branched processes. Both cells are monostratified in the same IPL sublayer (see 90° rotation). (B) Two more monostratified ACs from a different wild type into wild type chimera. (C) A large-field symmetrical AC derived from a lak mutant develops normally in a wild-type host retina. A 90° rotation shows that its arbors are monostratified. (D-F) Projection errors of ACs situated in lak mutant retinas. A wild-type-derived AC in a lak mutant retina (D) projects diffusely into the IPL, resembling the projection errors (arrows) made by lak mutant ACs in mutant retinas (E,F). (G-J) Sections through chimeric retinas, cut perpendicular to the IPL. When the host retina is wild type (G,H), donor-derived GFP+ AC processes are confined to specific sublayers (1), regardless of whether the donor is wild type (G) or lak mutant (H). Wild-type ACs in a lak mutant retina (I) show local perturbations of sublaminar targeting, similar to the phenotype of lak-into-lak chimeras (J). Arrow 1, normal stratification; arrow 2, disrupted stratification.

View larger version (23K): [in a new window]   Fig. 8. Model of IPL formation and sublamination in wild-type and lak mutants. (A-C) IPL formation in wild-type retina. At 2 dpf (A), ACs (green, blue) start to grow neurites. GCs and/or their dendrites (red), which have already grown out to form a proto-IPL, provide a signal that either orients ACs and their processes towards the nascent IPL (model 1, left), or else stabilizes IPL-oriented processes (model 2, right). At 2.5 dpf (B), ON (blue) and OFF (green) ACs have ramified their dendrites within the IPL (gray shaded area), but separate sublayers are not yet distinguishable. ON and OFF IPL domains (light blue and green shading) may already be distinct, but so close together within the developing IPL that they look like a single diffuse layer (model 1, represented by the two left-hand cells in B). Alternatively, ON and OFF strata may not be separate yet at this stage, in which case ACs would project diffusely throughout the extent of the IPL (model 2; right-hand cells of B). By 5 dpf (C), distinct ON and OFF sublayers are clearly evident. GCs are shown in pink; their dendrites are not fully depicted in B or C. (D-F) IPL formation in the absence of GCs. In lak mutants at 2 dpf (D), ACs lack the orienting signal from GCs and grow neurites in random directions, or towards each other. Their cell bodies are often ectopically positioned adjacent to the ILM, which causes the early IPL to form there as well. By 2.5 dpf (E), the tendency of AC neurites to grow towards each other has led to formation of an IPL. Because of the initial disorganization of AC somata and neurites, however, the nascent IPL is uneven. AC somata continue to accumulate between the IPL and the ILM, forming a layer of `misplaced' ACs. By 5 dpf (F), much of the unevenness has dissipated and IPL sublayers have formed, indicating that there are cues available to ACs that allow correction of early errors. One such mechanism may be homotypic attraction between ACs. However, these cues are not sufficient to allow correction of every error, leading to local perturbations of IPL sublamination (arrow).