Inhibitory neuron migration and IPL formation in the developing zebrafish retina

The mature vertebrate retina is a highly ordered neuronal network of cell bodies and synaptic neuropils arranged in distinct layers. Little, however, is known about the emergence of this spatial arrangement. Here, we investigate how the three main types of retinal inhibitory neuron (RIN) – horizontal cells (HCs), inner nuclear layer amacrine cells (iACs) and displaced amacrine cells (dACs) – reach their specific laminar positions during development. Using in vivo time-lapse imaging of zebrafish retinas, we show that RINs undergo distinct phases of migration. The first phase, common to all RINs, is bipolar migration directed towards the apicobasal centre of the retina. All RINs then transition to a less directionally persistent multipolar phase of migration. Finally, HCs, iACs and dACs each undergo cell type-specific migration. In contrast to current hypotheses, we find that most dACs send processes into the forming inner plexiform layer (IPL) before migrating through it and inverting their polarity. By imaging and quantifying the dynamics of HCs, iACs and dACs from birth to final position, this study thus provides evidence for distinct and new migration patterns during retinal lamination and insights into the initiation of IPL formation.


Fig S2: Models of IPL formation discussed in recent literature
A. 1. RGCs and ACs migrate in an overlapping sequence, resulting in interdigitated cell bodies. 2. The initiation of the IPL by RGC dendrites splits the iACs and the dACs. 3. ACs then direct their neurites towards RGC dendrites regardless of somal position. 4. RGCs and ACs stratify. 5-6. BCs direct their processes towards the IPL and stratify. B. 1. ACs arrive at a pre-formed RGC layer. 2. iACs and dACs initiate IPL formation by directing processes towards each other. 3. RGC dendrites accumulate beneath dACs 4. RGC dendrites bypass cell bodies of dACs and joins up with the iAC and dAC derived plexus. 5-6. BCs stratify after RGCs and ACs stratify.  Example of iAC (white) migrating tangentially about half a somal length via a process extending laterally from the basal side of the cell. B. Example of a dAC (white) migrating tangentially via process extending from its flattened soma as it migrates basally into the GCL. Times are shown in h:min relative to the start of the movies. Movies start at ~48hpf.

Fig S5: Example of an atypical dAC migrating into the GCL
Selected frames from a movie of a dAC (orange arrowheads) migrating into the GCL later than usual, when RGCs in the same clone are already clearly stratified. The dAC does not flatten, but squeezes through the forming IPL rapidly. For comparison, a dAC that migrated earlier is also indicated (blue arrowheads). Movie starts at ~48 hpf.

Fig S6: iACs and dACs differentially stratify in the proto-IPL
A. Isolated labelled dACs identified in 48-60 hpf SoFa2 retinas, where RINs are mosaically labelled with Ptf1a driven YFP, BCs are labelled using Crx driven CFP and RGCs are labelled using Ath5 driven RFP. Each column shows a different presumptive dAC. Images are arranged from left to right to suggest a possible sequence of migration. YFP labelled cells found basal to the BC plexus generally run lateral processes across the interface of the BC (cyan) plexus and the RGC plexus (magenta). B. Most isolated labelled iACs identified in the SoFa2 retina appear to initially stratify in the apical side of the BC plexus. Images are arranged from left to right to suggest a possible sequence of migration. C. Rarely, iACs appear to stratify in the middle of the BC plexus. Images are arranged from left to right to suggest a possible sequence of migration. D. The SoFa2 retina for starburst ACs using Sox2 antibody. A starburst iAC and a starburst dAC close to each other are seen to stratify on the apical side of the BC plexus, and at the interface between the BC plexus and RGC plexus, respectively.

Supplementary Movie 1: RINs may switch from bipolar migration to multipolar migration
This movie shows the retina of a Ptf1a:DsRed embryo that was injected with GCaMP-NLS mRNA at the one cell stage. Imaging commenced ~45 hpf. The movie first runs through the z stack in the first time point, and it can be seen that RINs (magenta) in more apical regions of the retina appear to have elongated, smooth morphologies with apical processes (coloured arrows) attached to the apical surface of the retina (outside white line). GCaMP-NLS labels most cell nuclei (green), and it can be seen that RINs gather in a layer in the middle of the retina that is two to three cell somas thick. The movie then switches to extended focus and progresses forward in time, and it can be seen that, out of the RINs that turn on DsRed while they are in more apical regions of the retina, all of them retract their apical processes only when they have reached the middle of the retina. It is difficult to see the morphology of most RINs located in the middle of the retina due to density of labelling. However, those that can be distinguished from nearby RINs appear multipolar (coloured open circles). Multipolar RINs located in the apical region of the retina (coloured closed circles) are not apparent until RINs have spent some time gathering in the middle of the retina, and appear to be cells that are migrating apically. These cells are likely HCs, and some of them (eg. purple closed circle) can be seen to divide.

Supplementary Movie 2: RINs transition to bipolar morphologies immediately after birth
Cells are best visualized by going through the z-stack slice by slice. This movie shows how Fig 2D was made. The left-most panel shows the z-projection with both the magenta (Ptf1a) and green (Ath5) channels, the second left panel shows the single z slice with both channels, the third panel shows the single z slice with the magenta (Ptf1a) channel only (overexposed in order to see the position of the apical process), the forth panel shows the Illustrator trace of the cell in that slice, and the fifth and right-most shows the cumulative traces. Time is shown as hours from cell birth. Z-slices are spaced 1µm apart.  Figure 4B captured in a Ptf1a:Gal4::UAS:YFP embryo. Time is written as h:min. In the left panel, the cell of interest has been pseudocoloured white using Photoshop. In the right panel, the uncoloured image is shown. The movie pauses at various timepoints and goes through the z-stack slice by slice. Z-slices are spaced 0.75µm apart. Movie starts at ~48 hpf.