XB-ART-17236J Neurobiol January 1, 1997; 32 (1): 45-61.
Xenopus Pax-6 and retinal development.
We have cloned a homolog of Pax-6 in Xenopus laevis. Its deduced amino acid sequence has a 95% overall identity with Pax-6 homologs in other vertebrates. It is expressed early in development in cells fated to form the eye and parts of the forebrain, hindbrain, and spinal cord. It has two phases of expression in the eye. In the early phase, from stage 12.5 to stage 33/34, Xenopus Pax-6 is expressed throughout the developing retina. In the late phase, after stage 33/34, it is excluded from mature cells in the outer half of the retina and from cells in the ciliary marginal zone, remaining only in amacrine and ganglion cells. Misexpression of Pax-6 early in development results in axial defects, but no specific eye phenotype is observed. Targeted misexpression in the retina at later stages does not result in any significant bias toward formation of amacrine or ganglion cells or away from photoreceptors. Ectopic expression of the proneural gene NeuroD alters the pattern of Pax-6, substantially reducing its expression in the eye field and later reducing or eliminating the eye itself. Our results show that Pax-6 expression appears to be necessary, but not sufficient, for eye formation in Xenopus.
PubMed ID: 8989662
Article link: J Neurobiol
Genes referenced: dnai1 frzb2 myc neurod1 pax6 rpe sult1a1
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|Figure 1 Structure and cDNA sequence of Xenopus Pax-6. (A) The complete DNA sequence of pPax-6 (above) and the deduced amino acid sequence (below). The two alternative initiation methionines are boxed. The paired domain is underlined with a single thin line, the paired homeobox is underlined with a double thin line, and the PST-rich region is underlined with a single thick line. (B) A diagram of the coding region of Pax- 6 showing the paired domain ( light gray), the paired homeodomain (medium gray), and the PST-rich domain (dark gray). The numbers above the diagram refer to the beginning and end of each of the three domains, as calculated from the nucleotide sequence in panel (A). The numbers below the diagram refer to the percent amino acid identity which the indicated species’ Pax-6 homolog shares with Xenopus over the length of the indicated domains. Numbers in parentheses show the percentage of amino acids in the PST-rich domain which are either proline, serine, or threonine. In Xenopus Pax-6 that number is 45%.|
|Figure 2 Expression of Pax-6 in the developing brain. In all panels except (E,F), anterior is to the right. In all panels except (A), (C), and (H), dorsal is up; in (A,C,H), dorsal is facing the viewer. (A) The earliest apparent staining pattern of Pax-6 at the neural plate stage (stage 14); arrow indicates the medial stripe. (B,C) A lateral (B) and dorsal (C) view of a stage 23 embryo. The arrowhead indicates the optic vesicle in (B), while the arrows indicate the two rhombomere-specific stripes in the hindbrain in (C). The midbrain staining is visible in (C) medial to the dotted white lines. (D) A lateral low-power view of a stage 28 embryo. The arrowhead indicates the gap in Pax-6 expression in the mesencephalon (optic tectum). The arrow points out the staining in the presumptive oculomotor nucleus. The dotted white line serves to distinguish the eye from the telencephalon/diencephalon. The developing eye also shows Pax-6 expression. (E) A transverse section of a stage 28 embryo at the level of the diencephalon: the arrow indicates staining in the dorsal diencephalon. (F) A transverse section of a stage 28 embryo through the spinal cord. The arrow indicates Pax-6 staining in the ventricular zone. N notochord. (G) A lateral view of a stage 42 embryo. The arrowhead shows a decrease of Pax-6 expression in the spinal cord, while the arrow shows a decrease of expression in the outermost layer of the retina. (H,I) A dorsal (H) and lateral (I) view of a stage 42 brain dissected free of surrounding tissue. The arrowhead in (H) points to staining in the olfactory bulb, while the arrows point to the rhombomere-specific staining. Abbreviations in (I): My meyelencephalon, Me mesencephalon, Di diencephalon, Te telencephalon. Scale bar is 200 mm in all except (E,F) (100 mm).|
|Figure 3 Pax-6 expression throughout retinal development. In all panels except (A), the vitreal surface of the retina is to the right. In all panels dorsal is up. (A) A sagittal section through a stage 14 embryo. Anterior is to the right. Note that the staining is in the neuroepithelial (Neu.) layer and not in the underlying mesoderm (Mes.). (B) A transverse section through a stage 28 optic cup. The arrow points to the presumptive lens epithelium, which expresses high levels of Pax-6 at this stage, while the arrowhead indicates the epidermis. Note that expression in the retinal neuroepithelium (NR) is essentially uniform at this stage. RPE retinal pigment epithelium. (C) A transverse section through a stage 33/34 retina. The intensity of the Pax-6 staining is decreasing in the outer nuclear layer (O). Inner nuclear layer I, ganglion cell layer G, lens Le. The ciliary marginal zone (CMZ) is outlined. (D) A transverse section through a stage 42 retina using DIC optics. The outer nuclear layer and the outer part of the inner nuclear layer (arrows) do not express Pax-6, nor does the CMZ (outlined). The lens at this stage has also turned Pax-6 off except in the most distal region. (E) A higher magnification of the boxed region in (D). The undifferentiated mitotic cells (arrowhead) of the CMZ (outlined in black) do not express Pax-6; neither do the photoreceptor cells of the outer nuclear layer (arrow). (F) Antibody labeling of Pax-6 in a stage 42 retina. The fluorescence is confined to the nuclei of the ganglion cells (G) and the inner nuclear layer (I). The outer part of the inner nuclear layer shows no Pax-6 staining (arrows); neither does the marginal zone (outlined in white). The fluorescence in the lens is nonspecific. Scale bar is 100 mm in panel (A), 50 mm in (B,C), and 20 mm in (D–F).|
|Figure 4 Examples of Pax-6 RNA injected embryos. In panels (A–C), anterior is to the right and dorsal is up. (A) An antisense injected embryo at stage 42 appears normal. It is representative of the mean DAI value for antisense RNA injection: DAI value = 5. (B) A sense injected embryo at control stage 42. The tail is shortened and bifurcated, but the anterior structures, including eye (arrow) and cement gland (arrowhead), appear to be normal. It is representative of the mean DAI value for sense RNA injection: DAI value = 7. (C) A sense injected embryo displaying a severe phenotype. Tail structures are absent and the neural tube has failed to close (arrow). The only recognizable anterior structure is the cement gland (arrowhead). DAI value = 10. (D) Summary of results from Pax-6 RNA injection experiments. S.E.M. = standard error of the mean. Scale bar = 200um|
|Figure 5 An analysis of Pax-6 myc DNA misexpression in vivo. In panels (B,C,F), the vitreal surface of the retina is to the right. In all panels dorsal is up. (A) A column graph showing the fraction of retinal cells expressing either Pax-6 (P6mycS) or control (pCS2 / MT) DNA. The error bars give the positive standard error of the mean (S.E.M.). GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer. (B) Fluorescent image of retinal cells expressing the pCS2 / MT DNA and stained for the c-myc epitope (green). Expression can be seen in all three retinal layers, particularly in the outer nuclear layer (arrowhead), and the staining fills the entire cytoplasm of the cell. (C) Fluorescent image of retinal cells expressing P6mycS DNA stained for the c-myc epitope (green). Expression can be seen in all three cell layers, including the outer nuclear layer (arrowhead), and the staining is confined to the nucleus. (D,E) Cells in the brain expressing Pax-6 myc protein stained Hoechst as a nuclear marker (D) or with the anti-myc antibody to showPax-6 expression (E). The cells expressing P6mycS localize it to their nuclei (arrowheads), although all nuclei in this section express the protein (arrow). (F) Cells in the outer nuclear layer (arrow) expressing P6mycS (anti-myc in green) still express rod opsin (anti-rod opsin in red), a photoreceptor-specific marker. Scale bar = 40 mmin panels (B,C) and 30 mm in (D–F).|
|Figure 6 NeuroD misexpression disrupts Pax-6 expression. All embryos were injected with NeuroD on the left side. In all panels dorsal is up. (A) Stage 14 neural plate stage, viewed anteriorly. The arrowhead shows the loss of the medial stripe of Pax-6 expression. Expression in the anterior crescent is reduced, but not eliminated (arrow). (B) A stage 23 embryo, viewed anteriorly. The area on the embryo’s left, where the eye should be, is outlined in black. The arrow indicates the forebrain staining on the injected side, which is largely unaffected. (C) A transverse section through a stage 26 embryo at the level of the eye. The staining in the brain on the injected side is reduced but not eliminated (arrow). Note the absence of an eye on the injected side (region outlined in black). Pax-6 expression is still visible in the presumptive lens epithelium (PLE) on the uninjected side (arrowhead). Scale bar 200 mm.|