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First published online 23 January 2008
doi: 10.1242/dev.011932

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meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye

José Bessa^1,*, Maria J. Tavares^1,*, Joana Santos^1,*, Hiroshi Kikuta², Mary Laplante², Thomas S. Becker², José Luis Gómez-Skarmeta¹ and Fernando Casares^1,3,

¹ CABD, CSIC-Universidad Pablo de Olavide, 41013 Seville, Spain.
² SARS Institute, N-5008 Bergen, Norway.
³ IBMC, Universidade do Porto, 4159-180 Oporto, Portugal.

View larger version (94K): [in this window] [in a new window]   Fig. 1. meis1 retracts accompanying the ath5 wave and becomes restricted to the CMZ. (A-D) meis1 and (E-H) ath5 expression analyzed by single in situ hybridization. Developmental stages are indicated as hours post-fertilization (hpf) at 28.5°C. Lateral views of whole-mount (A,E) or dissected (B-D,F-H) eyes, with dorsal up and anterior to the left. The front of the ath5 domain is marked by black arrowheads. The red arrowhead in D points to meis1 expression in the prospective ciliary margin. (I-M) Transverse 40 µm vibratome sections. (I,J) Dorsal is up. meis1 is weakly expressed in the lens ectoderm before its thickening (I), but no signal is detected once the lens placode is formed (J). (K-M) Dorsal is to the left. meis1 and ath5 expression domains are complementary as shown by double in situ hybridization (K,L). Approximate limits of the ath5 signal are indicated by the black arrowheads. (M) At 42hpf, meis1 expression is detected by in situ hybridization in the ciliary margin (red arrowheads) and in the postmitotic ganglion cells (black arrowhead). L, lens.

View larger version (61K): [in this window] [in a new window]   Fig. 2. meis1 is required for the growth of the eye primordium. (A,B) Lateral views of representative 72hpf control-MO (A) and meis1-MO (B) -injected fish. meis1 morphants are microphthalmic. (C,D) Confocal images of dissected eyes stained for propidium iodide (nuclei), rhodamine-phalloidin (filamentous actin) and Islet1, which labels GCL nuclei and some in the INL. The reduced eyes from meis1 morphants show apparently normal retina lamination (D,D'), but fewer cells than control eyes (C,C'). Area (E) and estimated volume (F) of control-MO and meis1-MO-injected embryos at 72hpf. meis1- morphant embryos show a significant (P<0.001) reduction in eye area and volume (45% and 60%, respectively). n=20 for each condition.

View larger version (70K): [in this window] [in a new window]   Fig. 3. meis1 is required for the G1-S transition and the expression of the G1-S regulators cyclin D1 and c-myc. (A-D) Histograms displaying DNA content (cell cycle profile) of cells from dissected 19hpf eyes of meis1-MO treated embryos relative to (A) controls (Crtl-MO), (B) meis1-MO+GFP-meis1 mRNA, (C) meis1-MO+cyclin D1 mRNA, and (D) meis1-MO+c-myc mRNA injected embryos. Number of replicates (n) and P values are indicated. meis1 knockdown induces a delay in G1, which is partially rescued by co-injection of GFP-meis1 (B), cyclin D1 (C) and c-myc (D) mRNAs. (E) Average percentages are shown for G1, S and G2/M DNA content. The cell-cycle profiles of control morphants and of uninjected, wild-type embryos are indistinguishable (not shown). (F-I) In situ analysis of cyclin D1 and c-myc transcription in control (F,H) and meis1 morphants (G,I) at 19hpf. meis1 morphants show a dramatic reduction in cyclin D1 and c-myc in the eye (red arrowheads). cyclin D1 and c-myc are still detected in other body regions (black arrowheads). Hybridization and reaction development were performed strictly in parallel. Representative embryos are shown.

View larger version (107K): [in this window] [in a new window]   Fig. 4. Clonal overerexpression of meis1 in the developing eye prevents differentiation and results in cell sorting. Single optical sections from confocal z-stacks of GFP (A-C) or GFP-Meis1 (D-F) expressing clones induced genetically in developing eyes. GFP-meis1 signal is nuclear. At 24-30hpf, both clone types frequently span the whole width of the neuroepithelium (A,D). Confocal optical sections through the central retina (B,E) and z-sections (C,F) of 48hpf eyes. At this stage, GFP clones comprise both Islet1-expressing and non-expressing cells (B,C). By contrast, same-stage GFP-Meis1 clones in the central retina do not contain Islet1-positive cells (E). GFP-Meis1 clones are often located in the CMZ (F). The arrowheads (C,F) point to the CMZ, and the retina and the lens (L) are outlined.

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