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Wilson JM
,
Sato K
,
Chernoff EA
,
Belecky-Adams TL
.
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Vertebrate homologues of musashi have recently been referred to as neural stem cell markers because of their expression patterns and RNA-binding interactions. In the context of the notch signaling pathway, Musashi-1 (Msi-1) is a regulator of neural cell generation, cooperating with notch to maintain mitosis. In an effort to identify definitive stem cell markers of the neural retina, a portion of the Msi-1 cDNA was cloned, and the expression of Msi-1 in the chick eye was analyzed. Using an Msi-1-specific antibody and RNA probe, we show that expression of Msi-1 in the early neural tube is consistent with neural stem identity. In the neural retina, expression starts shortly before embryonic day 3 (E3) and continues up to and including E18. A BrdU incorporation assay shows Msi-1 to be found in both proliferating and differentiating cells of E5 neural retina. At E8 (when proliferation is complete in the fundus of the retina) and E18 (mature retina) Msi-1 expression was found in the ciliary marginal zone (CMZ) as well as in a subpopulation of differentiated cells, including photoreceptors and ganglion cells.
Fig. 1. Msi-1 amino acids are highly conserved across species. Full-length sequences were analyzed using ClustalW to compare homology of the chick Msi-1 and human, mouse, Xenopus laevis and Drosophila. Black boxes indicated amino acids that are conserved in the chick. Dashes indicate regions where sequences of one species were not found in other species. The region of the protein encoded by the in situ hybridization probe is shown as underlined, bold and italicized letters.
Fig. 2. Msi-1 is enriched in the chick nervous system and sensory placodes. Wholemount in situ hybridization with HH st12-14 (A and C) and 18-21 (D–F, H) embryos and section in situ hybridization using cryosections from HH st12 and HH st21 (B, G, I, and J) were performed to determine where Msi-1 was expressed. (A) At HH st14 embryos treated with anti-sense probe, Msi-1 was confined to the developing nervous system (arrows) and otic placode (arrowhead). Signal was not apparent in the optic vesicle. (B) The results of the whole embryo in situ hybridization were verified by in situ hybridization using frozen sections. The cross section shown in A at the level of the hindbrain and otic vesicles show signals in the neural tube (arrow) and otic vesicles (arrowhead). The strength of the signal is considerably less in the roof plate in comparison to the rest of the neural tube. (C) HH st13 embryos treated with a control sense Msi-1 probe showed no apparent signal. (D) A low power magnification of an HH st19 embryo incubated with antisense Msi-1 probe showed signal enriched in the nervous system, similar to earlier stages. (E) Higher magnification view of the craniofacial region of embryo, shown in (D), showed strong signal within the forebrain and the optic cup (OC) and lightly in the pharyngeal arches (arrows). (F) Strong expression in the HH st21 spinal cord was best seen in a dorsal view of labeled embryos. (G) Signal obtained with wholemount in situ hybridization was checked using sections from HH st21 embryos. Heavy signal was detected in the forebrain, optic cup, lens vesicle, and nasal placode. (H) HH st18 embryos incubated with control sense probe show a small amount of trapping artifact in the optic cup. (I and J) Sections incubated with control sense probe show no apparent signal. D, dorsal; V, ventral; OC, optic cup; HL, hindlimb; NT, neural tube; NP, nasal placode; rp, roof plate. Magnification bar in (A)–(H) equals 500 μm.
Fig. 3. Msi-1 is spatiotemporally regulated in the developing neural tube and optic cup. (A) Comparison of expression patterns of a known midbrain–hindbrain marker, Pax2, and Msi-1 in whole chick embryos at HH st12. Lines represent sections taken in ensuing panels. (B–D) A series of sections from anterior to posterior regions of an HH st12embryohead were immunolabeled with Msi-1 antibody. The midbrain–hindbrain region (C) appears to have more label than the forebrain (B) and caudal hindbrain (D). (E) Immunolabel of coronal sections through and E4 eye showed heavy label in the fundus of the retina, and decreasing amounts of signal towards the periphery. Signal was also detected in the ectoderm and the lensepithelium adjacent to the ectoderm (inset E). (F and G) Low Msi-1 signal was detected at the vitreal edge of the fundus in E6 retina (F), while peripheral regions were heavily labeled (G). (H) Immunolabel of cryosections through the forebrain showed consistent label at the ventricular edge of the E6 proliferating neural tube. (I–K) Msi-1 immunoreactivity in cryosections from E8 embryos was similar to that seen at E6, with stronger label detected in peripheral retina, CMZ and ciliary epithelium (J and K) than in the fundus of the retina (I). However, signal in the putative ganglion cell, inner and outer nuclear layers became apparent at this stage (I). (L–N) Protein signals could be detected in the outer nuclear (ONL) and ganglion cell layers (GCL), as well as the vitreal half of the inner nuclear layer (INL) of E18 retina. Immunolabel of E18 retina showed no fundal to peripheral gradient in the retina, however the layer-specific labeling noted at E8 was still apparent (L and M). There was continued labeling of the CMZ and ciliary epithelium at E18 (N). (E–G, I–M) Arrows point to RPE where there is no apparent signal (E). INL, inner nuclear layer; ONL, outer nuclear layer; GCL, ganglion cell layer; RPE, retinal pigmented epithelium; CMZ, ciliary marginal zone; V, vitreal. Magnification bar in (A)–(N) equals 100 μm.
Fig. 4. Msi-1 label is found in proliferating and differentiating cells of the retina and in the CMZ. (A–C) Embryos were treated with bromodeoxyuridine (BrdU) for 5 h, fixed, and cryosections through the fundus of the retina triple-labeled with antibodies that recognize BrdU (A), Msi-1 (red; B), and Hoechst dye for DNA (blue in side panel, B). Arrows indicate a cell that is BrdU (−) (A) and Msi-1 (+) (B). Superimposed images show three types of labeled cells (Msi-1+/BrdU−, Msi-1+/BrdU+, and Msi-1−/BrdU+) (C). (D–F) Cells of the CMZ were double-labeled with transitin (D) and Msi-1 (E). Brackets show overlapping expression patterns in the CMZ and ciliary epithelium indicating Msi-1 is present in retinal precursors. Magnification bar in (A) equals 100 μm.
Fig. 5. Msi-1 is co-expressed with markers specific for differentiating cells of the retina. (A–L) Cryosections from E8 embryos were double-labeled with Msi-1 antibody (A, D, G, and J) and visinin (photoreceptors; B), HuC/D (ganglion cells; E), Brn3a (subset of ganglion cells; H), vimentin (Mueller glia; K), Double-labeled cells are shown in the third panel for each antibody (C, F, I, and L). Magnification bar in (A) equals 100 μm.
Fig. 6. Msi-1 is not expressed in RPE and subpopulations of bipolar cells and horizontal cells. (A–L) Cryosections from E4 and E8 embryos show where Msi-1 expression is absent. (A–C) Prox1 (horizontal cells; B) did not detect the same E8 retinal cells as Msi-1 (A) indicated in the overlay (C). (D–F) Chx10 (bipolar cells and subsets of undifferentiated cells; E) did not detect the same populations of E8 retinal cells as Msi-1 (D) indicated here when the images were superimposed (F). (G–L) DIC images of central E8 (H) and central E4 (K) show an intrinsic marker of pigment in the RPE (arrow), however, RPE cells, E8 Msi-1 label (G) and E4 Msi-1 label (J) do not overlap as indicated when the respective images are merged (I and L). Magnification bars in (A), (D), (G) and (J) equal 100 μm.
