Chang WS and Harris WA (1998), Sequential genesis and determination of cone an...

XB-ART-14867

J Neurobiol 1998 Jun 01;353:227-44.

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Sequential genesis and determination of cone and rod photoreceptors in Xenopus.

Chang WS , Harris WA .

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In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development.

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Species referenced: Xenopus laevis
Genes referenced: calb1 opn1lw

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	Figure 1 Temporal and spatial pattern of photoreceptor specification in Xenopus. Sections through the central retina of embryos at progressive stages of development were stained with antibodies to calbindin (A,C,E,G), rod opsin (B,D,F), or both markers (H). (A,B) At stage 33/34, a number of cells at the scleral edge of the central retina express calbindin [arrows in (A)], while only a single cell in the dorsal retina is labeled with anti-rod opsin [arrow in (B)]. (C,D) By stage 35/ 36, the spatial extent of labeling with either marker has expanded. Scattered cells expressing calbindin (C) appear in more dorsal locations than cells labeled with anti-rod opsin (D). (E,F) At stage 37/38, many more cells have been recruited into the developing photoreceptor layer. Calbindin-expressing cells (E) consistently appear in more peripheral locations than rod photoreceptors (F), which are notably absent from the ventral retina. (G) In a stage 46 retina, the specific localization of calbindin in cones is clearly revealed. Note the distinctive tapered outer segments and oil droplets of the labeled cells. Unlabeled rod outer segments are indicated (asterisks). (H) In a section of a stage 37/38 retina stained with both anti-calbindin (red) and anti-rod opsin (green) antibodies, no cells are double-labeled, confirming that calbindin is specifically expressed in cones. Scale bar Â 50 mm for (AâF) and 20 mM in (G,H). L Â lens. (AâF) Dorsal is to the left.
	Figure 2 Nucleotide and deduced amino acid sequence of Xenopus red cone opsin, and alignment with opsins from other species. (A) The open-reading frame of the cone opsin cDNA was determined by complete sequencing of pXCOP 2.4. Stop codons in untranslated regions are indicated with asterisks. (B) Alignment of amino acid residues representing the predicted transmembrane domain VII of Xenopus red cone opsin with the same regions of red- and greensensitive opsins from chicken and goldfish. Boxed amino acid residues are conserved in all vertebrate long-wavelength-sensitive opsins. Extensive homology with other red opsins in this region confirms that the Xenopus cDNA encodes a cone opsin pigment with maximal sensitivity in the long-wavelength range. The lysine (K) residue which functions as the retinal chromophore attachment site in all opsins is indicated in bold.
	Figure 3 In situ hybridization with opsin RNA probes for the major cone and rod subtypes in Xenopus. (A) Whole-mount staining of a stage 33/34 embryo with the red cone opsin probe shows expression in several scattered cells in the dorsal retina, and also in the pineal organ (Pi ) , which is slightly out of the focal plane. Dotted lines indicate the outline of the developing eye. (BâG) Double in situ hybridization of retinas at progressive stages of development with cone (purple) and rod (red) opsin probes. Boxed areas in (B,D,F) are shown at higher magnification in (C,E,G) . (B,C) At stage 33/34, approximately equal numbers of labeled cones and rods appear in the same section. Two cones [black arrows in (C)] and a single rod [white arrow in (C)] are strongly labeled in the dorsal-central retina. (D,E) At stage 35/36, additional cells begin to express opsin mRNAs, with more labeled cones than rods. Note that labeled cones and rods can arise in independent locations in the retina, and that cones are the most dorsally-located photoreceptors [arrows in (E)]. (F,G) By stage 37/38, many cells in the photoreceptor layer are expressing cone or rod opsin. At this stage, the numbers of labeled cones and rods are approximately equal. Scale bar Â 100 mm in (A), 50 mm in (B,D,F) , and 25 mm in (C,E,G) . L Â lens. (BâG) Dorsal is to the left.
	Figure 4 In situ hybridization of tangential retinal sections showing the planar organization of cones and rods. (A) Cells expressing red cone opsin are often surrounded by five or six unlabeled photoreceptors, but there is no rigid pattern with which the labeled cells are arranged. (B) Double in situ hybridization with cone (purple) and rod (red) opsin probes also shows that although photoreceptors are arranged with some degree of minimal spacing, the overall organization of the photoreceptor mosaic in Xenopus is imprecise. Some cells are not labeled by either probe ( asterisks ) and likely represent a different cone subtype. Scale bar Â 20 mm.
	Figure 5 Cone photoreceptor determination in isolated cell culture. (A,B) A field of three isolated cells from a dissociated retinal culture initiated at stage 24 and grown in vitro for 24 h. The coverslip was triple-labeled with mAB XAP-1 (red) , anti-calbindin (green) , and Hoechst nuclear stain (blue) . (A) One cell is labeled with XAP-1 (arrow) , and it displays a photoreceptor- like morphology, with a tapered process at one end. Unlabeled cells (arrowheads) have a more fibroblast-like appearance. (B) The same field, viewed with fluorescein optics, shows that the XAP-1/ cell is also stained with anti-calbindin, indicating that it developed as a cone photoreceptor. (C) A field of several cells from a culture initiated at stage 26 and processed for in situ hybridization with the red cone opsin probe. Two labeled cells are present in this field; one is isolated (arrow) , while another (arrowhead) is in contact with at least two other cells. Scale bar Â 25 mm in (A,B) and 10 mm in (C).
	Figure 6 Quantitative analysis of photoreceptor marker expression in isolated retinal cells from progressive stages of development indicates that cones are determined earlier than rods. Isolated cells expressed XAP-1 and calbindin starting at stage 24, while isolated rods did not appear until stage 26. None of the markers was detected in isolated cells from retinas dissociated prior to stage 24, indicating that this is the earliest stage of photoreceptor induction in vivo. The percentage of isolated cells expressing photoreceptor markers increased with developmental stage, although many more isolated cones were counted than rods. The plot represents the mean percentages of cells expressing photoreceptor markers in cultures of retina dissociated at the indicated developmental stages; for each time point, a minimum of eight coverslips were scored. Data from seven individual culture experiments were combined, with bars representing the mean error (S.E.M.) between different runs.
	Figure 7 The first cone photoreceptors are born before the first rod photoreceptors in Xenopus. The pattern of cone and rod genesis was examined by injecting embryos with BrdU at stage 22 (A,B) or stage 31 (C,D) , then examining the expression of cone (A,C) and rod (B,D) opsin RNA at stage 37/38. (A) In the central retina of an embryo injected with BrdU at stage 22, a single postmitotic cone ( asterisk ) and three BrdU-labeled cones (arrows) are indicated. (B) In contrast, rod opsin-expressing cells are all labeled with BrdU (arrows) . (C,D) In embryos injected at stage 31, more rods are postmitotic than cones. (C) Three cones are labeled with BrdU (arrows) , indicating that they underwent S-phase some time after stage 31. Two unlabeled cones are also indicated ( asterisks ) . Note the unlabeled cells just vitreal to the position of the cone nuclei, where rod cell bodies are located (r). (D) Only one rod nucleus is stained with anti-BrdU (arrow) , while three rods are unlabeled ( asterisks ) . A number of nuclei in more scleral positions show BrdU labeling (c); these likely represent later-born cone photoreceptors. PE Â pigmented epithelium. Scale bar Â 10 mm.
	Figure 8 Profile of cone and rod genesis during early stages of retinal development in Xenopus. Cones are the first photoreceptors to be born, since a few postmitotic cones were counted in embryos injected with BrdU at stage 22, while no rods were postmitotic at this stage. The first unlabeled rods appeared in embryos injected at 24, indicating that rod genesis initiates slightly later than cone genesis. Up to stage 28, a higher percentage cones are postmitotic than rods, but by stage 31, the is reversed, indicating that many rods are born between stages 28 and 31. For each time point, a mini- mum of 10 sections from three different embryos were scored. Error bars Â S.E.M.