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First published online 23 January 2008
doi: 10.1242/dev.012088

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Evidence for an evolutionary conserved role of homothorax/Meis1/2 during vertebrate retina development

Peer Heine, Eva Dohle, Keely Bumsted-O'Brien^*, Dieter Engelkamp and Dorothea Schulte

Department of Neuroanatomy, Max-Planck-Institute for Brain Research, Deutschordenstr. 46, 60528 Frankfurt, Germany.

View larger version (119K): [in this window] [in a new window]   Fig. 1. Spatial and temporal expression of Meis1 and Meis2. (A-C,E) Expression of Meis2 in chick at (A) HH7 (arrow, anterior neural plate), (B) HH15 (E2; arrow, eye cup), (C) HH19 (early E3) and (E) HH25 (E4.5). Meis2 expression is lost in RPCs after late E2. Arrows in C indicate the central domain, where Meis2 is downregulated. (D) Expression of Ath5 in chick at HH19. Arrows indicate where Ath5+ cells appear at HH19. (F-F'') Double immunostaining for Meis2 and Pax6 at HH11. (G-J) Expression of Meis1 in chick at (G) HH11, (H) HH15, (I) HH19 and (J) HH25. Arrow in J indicates Meis1 downregulation in the RGC layer. (K) Expression of Meis2 in mouse eye anlage at E9.5. (L) Meis2 is not expressed in the E12.5 mouse retina. (M,N) Meis1 expression in the mouse optic vesicle (M) and neural retina (N). Expression is downregulated in the RGC layer (arrow). Boxed areas in E and J are shown at high magnification in the insets. Le, lens; lp, lens placode; mes, mesencephalon; nR, neural retina; ov, optic vesicle; PE, pigmented epithelium; SE, surface ectoderm. Scale bars: 200 µm in C,D,I; 500 µm in B,E,H,J; 50 µm in K,M; 100 µm in L,N.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Forced expression of a dominant-negative Meis interferes with RPC proliferation. (A-C) MeisEnR-transfected chick eyes are microphthalmic. (D-E') BrdU incorporation following ectopic expression of GFP (D,D') or MeisEnR together with GFP (E,E'). (F) Quantification of the results from D-E'. Error bars indicate s.d. (G,H) TUNEL labeling of HH16 retinae transfected with nuclear red fluorescent protein (nRFP; G) or MeisEnR together with nRFP (H). (I) TUNEL labeling on a HH16 chick retina treated with DNase I to visualize the effectiveness of the labeling strategy. (J-L) Clonal analysis. Representative clones infected with RIS(B)-GFP (J), RIS(B)-Meis2 (K) and RIS(B)-MeisEnR (L) visualized with the virus-specific antibody p27 (green). RIS(B)-MeisEnR-infected cells express the Myc-tag C-terminally fused to EnR (red, L). (M) Number of cells per clone and per section plotted as a function of the frequency at which clones of the respective sizes were observed.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Expression of Meis-inactivating constructs reduces RPC proliferation and cyclin D1 expression. (A-E) HH15 chick eyes transfected with the listed expression constructs together with GFP (green) and stained for phosphorylated histone H3 (pH3, red). (A) Control siRNA construct containing a randomized targeting sequence. pH3+/GFP+ indicates percentage of transfected cells in mitosis. (F-J) HH15 chick eyes stained for cyclin D1 or cyclin D2 following transfection with the constructs indicated. (F,H) cyclin D1, (G,J) cyclin D2. (K-M) Retinae transfected with MeisEnR+GFP (K), GFP (L) or MeisEnR+GFP together with cyclin D1 (M), stained for GFP (green) and pH3 (red); cell nuclei are counterstained with DAPI (blue). The position of pH3-labeled cell nuclei is indicated in the right-hand and middle panels of K. (N) Percentage of cells in mitosis (y-axis) for each transfected construct (x-axis). (O-R) Expression of Ath5 (O,Q) following transfection with the constructs indicated. The boxed areas in O,Q are shown at high magnification in O',Q', respectively. F,G, H-J, O,P and Q,R show neighboring sections. (I,P,R) Transfection controls.

View larger version (108K): [in this window] [in a new window]   Fig. 4. Decrease in Pax6, Six3 and Chx10 expression following MeisEnR transfection. (A-H) Expression of Pax6 (A-D), Chx10 (E,F) or Six3 (G,H) in HH15 chick eyes transfected with control-EnR (A,C,E,G) or MeisEnR (B,D,F,H). (A,B,E,F) Wholemounts. (C,D,G,H) Vibratome sections. (I) Quantification of the gene expression changes observed. (J) RT-PCR of two independent experiments (Exp_a, Exp_b) for Pax6 and β-actin of chick HH15 eyes transfected with GFP (control) or M1si_I and M2si_I together (Msi). PCR conditions were: 30 seconds 94°C, 30 seconds 60°C, 1 minute 72°C for 27 cycles. (K) Relative pixel intensity of the ethidium bromide-stained bands from J (Pax6/β-actin). Simultaneous knock-down of Meis1 and Meis2 expression decreases Pax6 transcript levels in the eye. (L,M) Pax6 fails to rescue cyclin D1 expression in MeisEnR-transfected chick retinae. Arrows indicate domains of MeisEnR/Pax6 misexpression and reduced cyclin D1 expression. (M) cyclin D1 expression following transfection of MeisEnR together with Pax6. (N-P) Expression of cyclin D1 (N) or cyclin D2 (P) following Pax6EnR misexpression. (O,L) Transfection control. L,M and N-P are neighboring sections. (Q) Quantification of the observed gene expression changes from Fig. 3F-J and data not shown.