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Influences on neural lineage and mode of division in the zebrafish retina in vivo.

Poggi L, Vitorino M, Masai I, Harris WA - J. Cell Biol. (2005)

Bottom Line: Proc.Natl.This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, United Kingdom.

ABSTRACT
Cell determination in the retina has been under intense investigation since the discovery that retinal progenitors generate clones of apparently random composition (Price, J., D. Turner, and C. Cepko. 1987. Proc. Natl. Acad. Sci. USA. 84:156-160; Holt, C.E., T.W. Bertsch, H.M. Ellis, and W.A. Harris. 1988. Neuron. 1:15-26; Wetts, R., and S.E. Fraser. 1988. Science. 239:1142-1145). Examination of fixed tissue, however, sheds little light on lineage patterns or on the relationship between the orientation of division and cell fate. In this study, three-dimensional time-lapse analyses were used to trace lineages of retinal progenitors expressing green fluorescent protein under the control of the ath5 promoter. Surprisingly, these cells divide just once along the circumferential axis to produce two postmitotic daughters, one of which becomes a retinal ganglion cell (RGC). Interestingly, when these same progenitors are transplanted into a mutant environment lacking RGCs, they often divide along the central-peripheral axis and produce two RGCs. This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

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ath5:GFP is expressed in both mitotic and differentiating retinal progenitors. (A) 3D lateral view of an ath5:GFP transgenic retina at 32 h after fertilization, as seen in all subsequent time-lapse recordings performed in this study (the image is a combination of several stacks). The diagram in B shows the main developmental steps of one retinal neuroepithelial cell. The cell soma undergoes apico-basal interkinetic nuclear movements. Mitosis (M) occurs in proximity of the apical surface (top). (C) Time-lapse series showing an ath5:GFP progenitor dividing at the apical surface (dashed line). Dividing progenitors and daughter cells are highlighted in green or red. The long arrows point at the apical processes that connect the ath5:GFP retinal progenitors to the apical surface. Short arrows point to the apical process of a differentiating ath5-positive cell. This process can be seen retracting in the right-most panel. (D) A single retinal progenitor cell is labeled with GAP-mRFP. The time-lapse series shows that ath5:GFP appears (t = 0) when the cell soma is migrating toward the apical surface (ap). At t = 45 min, the cell divides and both daughter cells migrate basally. White arrows indicate the position of the cell soma of the progenitor or daughter cells. (E) Time-lapse sequence showing the sequential steps of RGC differentiation (also see the diagram in B). The cell highlighted in green is expressing ath5:GFP. At t = 0, the cell is connected to the apical and basal surface with its apical and basal processes, respectively. Later on, the apical process (ap and arrows) retracts from the apical surface (t = 1.50), and the axon starts to elongate and extend from the basal process (bp and arrows). (F–H) Confocal images of an ath5:GFP transgenic retina at 30 h after fertilization, hybridized with ath5 mRNA probe (in red). Each image is a projection of stacks. The white box indicates what is represented in G. Arrows point at two dividing progenitors at the apical surface that are expressing both ath5:GFP and ath5 mRNA, although clearly at different amounts. Arrowheads point at differentiating RGCs. (H) 3D representation of a dividing ath5:GFP cell (outlined) expressing ath5 mRNA. A, anterior; ap; apical process; bp, basal process; D, dorsal; P, posterior; RGC, retinal ganglion cell; RPI, retinal pigmented epithelium; V, ventral.
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fig1: ath5:GFP is expressed in both mitotic and differentiating retinal progenitors. (A) 3D lateral view of an ath5:GFP transgenic retina at 32 h after fertilization, as seen in all subsequent time-lapse recordings performed in this study (the image is a combination of several stacks). The diagram in B shows the main developmental steps of one retinal neuroepithelial cell. The cell soma undergoes apico-basal interkinetic nuclear movements. Mitosis (M) occurs in proximity of the apical surface (top). (C) Time-lapse series showing an ath5:GFP progenitor dividing at the apical surface (dashed line). Dividing progenitors and daughter cells are highlighted in green or red. The long arrows point at the apical processes that connect the ath5:GFP retinal progenitors to the apical surface. Short arrows point to the apical process of a differentiating ath5-positive cell. This process can be seen retracting in the right-most panel. (D) A single retinal progenitor cell is labeled with GAP-mRFP. The time-lapse series shows that ath5:GFP appears (t = 0) when the cell soma is migrating toward the apical surface (ap). At t = 45 min, the cell divides and both daughter cells migrate basally. White arrows indicate the position of the cell soma of the progenitor or daughter cells. (E) Time-lapse sequence showing the sequential steps of RGC differentiation (also see the diagram in B). The cell highlighted in green is expressing ath5:GFP. At t = 0, the cell is connected to the apical and basal surface with its apical and basal processes, respectively. Later on, the apical process (ap and arrows) retracts from the apical surface (t = 1.50), and the axon starts to elongate and extend from the basal process (bp and arrows). (F–H) Confocal images of an ath5:GFP transgenic retina at 30 h after fertilization, hybridized with ath5 mRNA probe (in red). Each image is a projection of stacks. The white box indicates what is represented in G. Arrows point at two dividing progenitors at the apical surface that are expressing both ath5:GFP and ath5 mRNA, although clearly at different amounts. Arrowheads point at differentiating RGCs. (H) 3D representation of a dividing ath5:GFP cell (outlined) expressing ath5 mRNA. A, anterior; ap; apical process; bp, basal process; D, dorsal; P, posterior; RGC, retinal ganglion cell; RPI, retinal pigmented epithelium; V, ventral.

Mentions: Recent BrdU experiments describe ath5 expression as a post–S-phase marker of retinal progenitors (Masai et al., 2005). However, GFP driven by the ath5 promoter and enhancer is detectable before RGCs complete their differentiation and possibly before their final mitosis (Masai et al., 2005). Therefore, we used these transgenic ath5:GFP fish embryos to perform long-term time-lapse imaging of ath5:GFP-expressing retinal progenitors to see when in the cell cycle these cells began to express GFP. Embryonic retinas that were imaged in 15-min intervals with optical sections 0.5 μm apart were taken through a volume up to 50 μm in depth for a minimum of 10 h starting from ∼32 h after fertilization (Fig. 1 A). A schematic describing the main features of the developing retinal neuroepithelium is shown in Fig. 1 B. These image sets were reconstructed into 3D time-lapse videos. ath5:GFP cells could be clearly seen undergoing M phase of the cell cycle at the apical surface (Fig. 1 C and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200509098/DC1). In a few instances, we transplanted ath5:GFP blastomeres that had been injected with a membrane-targeted RFP construct (growth-associated protein [GAP]–mRFP) and used double labeling to highlight cells as they first turned on GFP. Although the RFP label was weak and faded rather quickly in these experiments, we were able to focus on dividing cells that became GFP positive during the recording sessions. In these cases, GFP first became visible in RFP-labeled cells as the nuclei of these cells were migrating toward the apical surface before metaphase (Fig. 1 D and Video 2). This apically oriented phase of interkinetic nuclear migration is generally associated with the G2 phase of the cell cycle (Seymour and Berry, 1975). Finally, ath5:GFP cells could be seen acquiring the anatomical characteristics of RGCs. Differentiation into RGCs is characterized by the retraction of the apical process, the migration of the cell body to the RGC layer of the retina near the vitreal surface, and the extension of a growth cone–tipped axon that grows along the vitreal surface toward the future optic nerve head (Fig. 1 E and Video 3).


Influences on neural lineage and mode of division in the zebrafish retina in vivo.

Poggi L, Vitorino M, Masai I, Harris WA - J. Cell Biol. (2005)

ath5:GFP is expressed in both mitotic and differentiating retinal progenitors. (A) 3D lateral view of an ath5:GFP transgenic retina at 32 h after fertilization, as seen in all subsequent time-lapse recordings performed in this study (the image is a combination of several stacks). The diagram in B shows the main developmental steps of one retinal neuroepithelial cell. The cell soma undergoes apico-basal interkinetic nuclear movements. Mitosis (M) occurs in proximity of the apical surface (top). (C) Time-lapse series showing an ath5:GFP progenitor dividing at the apical surface (dashed line). Dividing progenitors and daughter cells are highlighted in green or red. The long arrows point at the apical processes that connect the ath5:GFP retinal progenitors to the apical surface. Short arrows point to the apical process of a differentiating ath5-positive cell. This process can be seen retracting in the right-most panel. (D) A single retinal progenitor cell is labeled with GAP-mRFP. The time-lapse series shows that ath5:GFP appears (t = 0) when the cell soma is migrating toward the apical surface (ap). At t = 45 min, the cell divides and both daughter cells migrate basally. White arrows indicate the position of the cell soma of the progenitor or daughter cells. (E) Time-lapse sequence showing the sequential steps of RGC differentiation (also see the diagram in B). The cell highlighted in green is expressing ath5:GFP. At t = 0, the cell is connected to the apical and basal surface with its apical and basal processes, respectively. Later on, the apical process (ap and arrows) retracts from the apical surface (t = 1.50), and the axon starts to elongate and extend from the basal process (bp and arrows). (F–H) Confocal images of an ath5:GFP transgenic retina at 30 h after fertilization, hybridized with ath5 mRNA probe (in red). Each image is a projection of stacks. The white box indicates what is represented in G. Arrows point at two dividing progenitors at the apical surface that are expressing both ath5:GFP and ath5 mRNA, although clearly at different amounts. Arrowheads point at differentiating RGCs. (H) 3D representation of a dividing ath5:GFP cell (outlined) expressing ath5 mRNA. A, anterior; ap; apical process; bp, basal process; D, dorsal; P, posterior; RGC, retinal ganglion cell; RPI, retinal pigmented epithelium; V, ventral.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171316&req=5

fig1: ath5:GFP is expressed in both mitotic and differentiating retinal progenitors. (A) 3D lateral view of an ath5:GFP transgenic retina at 32 h after fertilization, as seen in all subsequent time-lapse recordings performed in this study (the image is a combination of several stacks). The diagram in B shows the main developmental steps of one retinal neuroepithelial cell. The cell soma undergoes apico-basal interkinetic nuclear movements. Mitosis (M) occurs in proximity of the apical surface (top). (C) Time-lapse series showing an ath5:GFP progenitor dividing at the apical surface (dashed line). Dividing progenitors and daughter cells are highlighted in green or red. The long arrows point at the apical processes that connect the ath5:GFP retinal progenitors to the apical surface. Short arrows point to the apical process of a differentiating ath5-positive cell. This process can be seen retracting in the right-most panel. (D) A single retinal progenitor cell is labeled with GAP-mRFP. The time-lapse series shows that ath5:GFP appears (t = 0) when the cell soma is migrating toward the apical surface (ap). At t = 45 min, the cell divides and both daughter cells migrate basally. White arrows indicate the position of the cell soma of the progenitor or daughter cells. (E) Time-lapse sequence showing the sequential steps of RGC differentiation (also see the diagram in B). The cell highlighted in green is expressing ath5:GFP. At t = 0, the cell is connected to the apical and basal surface with its apical and basal processes, respectively. Later on, the apical process (ap and arrows) retracts from the apical surface (t = 1.50), and the axon starts to elongate and extend from the basal process (bp and arrows). (F–H) Confocal images of an ath5:GFP transgenic retina at 30 h after fertilization, hybridized with ath5 mRNA probe (in red). Each image is a projection of stacks. The white box indicates what is represented in G. Arrows point at two dividing progenitors at the apical surface that are expressing both ath5:GFP and ath5 mRNA, although clearly at different amounts. Arrowheads point at differentiating RGCs. (H) 3D representation of a dividing ath5:GFP cell (outlined) expressing ath5 mRNA. A, anterior; ap; apical process; bp, basal process; D, dorsal; P, posterior; RGC, retinal ganglion cell; RPI, retinal pigmented epithelium; V, ventral.
Mentions: Recent BrdU experiments describe ath5 expression as a post–S-phase marker of retinal progenitors (Masai et al., 2005). However, GFP driven by the ath5 promoter and enhancer is detectable before RGCs complete their differentiation and possibly before their final mitosis (Masai et al., 2005). Therefore, we used these transgenic ath5:GFP fish embryos to perform long-term time-lapse imaging of ath5:GFP-expressing retinal progenitors to see when in the cell cycle these cells began to express GFP. Embryonic retinas that were imaged in 15-min intervals with optical sections 0.5 μm apart were taken through a volume up to 50 μm in depth for a minimum of 10 h starting from ∼32 h after fertilization (Fig. 1 A). A schematic describing the main features of the developing retinal neuroepithelium is shown in Fig. 1 B. These image sets were reconstructed into 3D time-lapse videos. ath5:GFP cells could be clearly seen undergoing M phase of the cell cycle at the apical surface (Fig. 1 C and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200509098/DC1). In a few instances, we transplanted ath5:GFP blastomeres that had been injected with a membrane-targeted RFP construct (growth-associated protein [GAP]–mRFP) and used double labeling to highlight cells as they first turned on GFP. Although the RFP label was weak and faded rather quickly in these experiments, we were able to focus on dividing cells that became GFP positive during the recording sessions. In these cases, GFP first became visible in RFP-labeled cells as the nuclei of these cells were migrating toward the apical surface before metaphase (Fig. 1 D and Video 2). This apically oriented phase of interkinetic nuclear migration is generally associated with the G2 phase of the cell cycle (Seymour and Berry, 1975). Finally, ath5:GFP cells could be seen acquiring the anatomical characteristics of RGCs. Differentiation into RGCs is characterized by the retraction of the apical process, the migration of the cell body to the RGC layer of the retina near the vitreal surface, and the extension of a growth cone–tipped axon that grows along the vitreal surface toward the future optic nerve head (Fig. 1 E and Video 3).

Bottom Line: Proc.Natl.This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, United Kingdom.

ABSTRACT
Cell determination in the retina has been under intense investigation since the discovery that retinal progenitors generate clones of apparently random composition (Price, J., D. Turner, and C. Cepko. 1987. Proc. Natl. Acad. Sci. USA. 84:156-160; Holt, C.E., T.W. Bertsch, H.M. Ellis, and W.A. Harris. 1988. Neuron. 1:15-26; Wetts, R., and S.E. Fraser. 1988. Science. 239:1142-1145). Examination of fixed tissue, however, sheds little light on lineage patterns or on the relationship between the orientation of division and cell fate. In this study, three-dimensional time-lapse analyses were used to trace lineages of retinal progenitors expressing green fluorescent protein under the control of the ath5 promoter. Surprisingly, these cells divide just once along the circumferential axis to produce two postmitotic daughters, one of which becomes a retinal ganglion cell (RGC). Interestingly, when these same progenitors are transplanted into a mutant environment lacking RGCs, they often divide along the central-peripheral axis and produce two RGCs. This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

Show MeSH
Related in: MedlinePlus