Intrinsic bias and lineage restriction in the phenotype determination of dopamine and neuropeptide Y amacrine cells

Abstract

Blastomere lineages are differentially biased to produce different neurotransmitter subtypes of amacrine cells (Huang and Moody, 1995, 1997,). To elucidate when this bias is acquired, we examined amacrine lineages at different early developmental times. Our experiments demonstrate that the bias to express dopamine and neuropeptide Y amacrine fates involves several steps before the formation of the definitive optic cup. At cleavage stages, a retinal progenitor that contributes large numbers of cells is already biased to produce its normal repertoire of dopamine amacrine cells, as revealed by transplantation to a new location, whereas the amacrine fate of a progenitor that contributes fewer cells is modified by its new position. At neural plate stages, not all retinal progenitors are multipotent. Nearly one-half populate only the inner nuclear layer and are enriched in amacrine cells. During early optic vesicle stages, an appropriate mitotic tree is required for dopamine and neuropeptide Y, but not serotonin, amacrine cell clusters to form. Thus, the acquisition of amacrine fate bias involves intrinsic maternal factors at cleavage, fate restriction in the neural plate, and specified mitotic patterns in the optic vesicle. At each of these steps only a subset of the embryonic retinal progenitors contributing to amacrine subtypes is biased; the remaining progenitors maintain multipotency. Thus, from the earliest embryonic stages, progenitors of the retina are a dynamic mosaic. This is the first experimental demonstration of amacrine fate decisions that occur during early embryonic periods in advance of the events described in the later, committed retina.

Fig. 1.

Diagram of the animal pole view of a 32-cell embryo. The five ipsilateral blastomeres that give rise to the retina are labeled with the Jacobson and Hirose (1981) nomenclature.Arrows demonstrate the blastomere transplantations that were performed: V1.2.1 to the position of D1.2.2 and D1.2.1 to the position of D1.1.2.

Fig. 2.

Changes in the neurotransmitter subtypes of amacrine cells descended from transplanted blastomeres.A, The number of large, bright 5-HT (LB-5HT) amacrine cells in a labeled blastomere's clone. Normally, V1.2.1 gives rise to a fewLB-5HT cells (V1.2.1N), but after transplantation to the D1.2.2 position (V1.2.1T), it assumes a quantitative fate more similar to that of the control blastomere of its new position (D1.2.2N).B, The number of DA amacrine cells in a labeled blastomere's clone. Normally, D1.2.1 gives rise to 32% of the DA cells in the retina, a fate it maintains when it is control-transplanted to its normal position in the embryo (D1.2.1C). It also maintains this DA amacrine fate when it is transplanted to the D1.1.2 position (D1.2.1T). D1.1.2, in comparison, gives rise to a very small number of amacrine cells [normal blastomere (D1.1.2N); control transplanted blastomere (D1.1.2C)]. Thus the DA amacrine fate of blastomere D1.2.1 is specified by the 32-cell stage.

Fig. 3.

Clones resulting from the intracellular labeling of single cells at neural plate stages. A, An embryo fixed 10 min after labeling. Only a single cell was injected with tracer (red). This sagittal section demonstrates that in the region of the eye field, the neural plate is approximately four to six cells thick and can be divided into an intermediate zone (i) and a deep zone (d). The superficial zone is comprised of non-neural ectoderm (e). B, A section through the stage 44 retina demonstrating a radial clone (redcolumn) containing cells in each retinal layer. This clone also contains a few dispersed cells, a configuration only observed in 3.3% of cases and consistent with reports in chick and mouse (Fekete et al., 1994; Reese and Tan, 1998). C, A layered clone that resides entirely in the INL. It contains cells in the outer sublamina and amacrine cells (arrows) in the inner sublamina. D, A layered clone that resides entirely in the outer sublamina of the INL. am, Inner sublamina of the INL; bh, outer sublamina of the INL;gc, ganglion cell layer; inl, inner nuclear layer; ph, photoreceptor layer. Scale bars, 100 μm.

Fig. 4.

The distribution of cells in clones descended from neural plate progenitors. A, The percentage of radial clones containing progeny in two nonadjacent layers (GC,BH; GC,PH; AM,PH), in three layers, or in four layers. B, The percentage of layered clones containing progeny in single layers (left side) or two adjacent layers (right side). In both categories, layered clones are found predominantly in the inner nuclear layer (BH; AM; AM,BH).

Fig. 5.

The spatial distribution of eye field progenitors that give rise to radial and layered clones. Both radial and layered progenitors are equally distributed between lateral (L) and medial (M) regions of the eye field. Radial progenitors are found slightly more frequently in intermediate (I, <40 μm) versus deep (D, >40 μm) cell layers, whereas layered progenitors are equally distributed throughout the depth of the neural plate.

Fig. 6.

Layered clone progenitors divide fewer times than do radial clone progenitors. The number of cell divisions that occurred after a neural plate progenitor was labeled was determined by the size of the clone. The percentage of clones in each size bin (1–5 cell divisions) is shown for layered clones confined to one layer (One/Layered), layered clones confined to two adjacent layers (Two/Layered), radial clones distributed to three layers (Three/Radial), and radial clones distributed to four layers (Four/Radial).

Fig. 7.

Layered clones are enriched in amacrine cells.A, Clones derived from eye field progenitors that contained more than one amacrine cell (AM) were analyzed as either radial (gray bars) or layered (black bars). The majority of clones containing small numbers of amacrine cells were radial, whereas nearly one-half of the clones containing large numbers of amacrine cells were layered.B, In layered clones, amacrine cells most frequently were siblings of other amacrine cells. They also were commonly siblings of bipolar cells (B), either alone or in common with horizontal (H) and Müller (M) cells. Rarely were amacrine cells only siblings of horizontal cells, and never were they siblings of only Müller cells.

Fig. 8.

Formation of DA and NPY amacrine clusters is inhibited when mitosis is repressed during optic vesicle stages.A, The percentage of DA amacrine cells found in clusters is completely inhibited when embryos are incubated in DNA replication inhibitors starting at stage 21. The percentage of clusters remains significantly repressed during optic vesicle stages (23–25) and gradually returns to nearly 50% of the normal level by optic cup stages (31). B, The percentage of NPY amacrine cells found in clusters is significantly repressed during optic vesicle stages (23–25) and returns to nearly 50% of the normal level by optic cup stages (31). C, The percentage of LB-5-HT amacrine cells found in clusters is reduced to slightly >50% during optic vesicle stages and returns to near normal by optic cup stages.St., Stage.