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Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye

Developmental Biology Programme, EMBL-Heidelberg, Meyerhofstr. 1, 69012 Heidelberg, Germany

View larger version (52K): [in a new window]   Fig. 1. Specific interference with gene translation using Six3 (MoSix3) and Pax6 (MoPax6) morpholinos. (A-F) Dorsal views of embryos at early somitogenesis stage (21); anterior is towards the left. (A,D) Embryos injected with control RNA at the one-cell stage show overall GFP expression. (B,C,E,F) At the two-cell stage, embryos were co-injected with the respective morpholinos and rhodamine dextran (RD) and analyzed using the GFP and rhodamine (Rhod) channel. mhb, mid-hindbrain boundary.

View larger version (113K): [in a new window]   Fig. 2. Six3 is required for retina and forebrain development. (A-E) Dorsal views of embryos (A,B) at six-somite stage (21) and (C-E) late gastrula stage (16), anterior is towards the left. (B) Injection of high MoSix3 concentrations results in loss of anterior structures (severe) compared with MoGFP control injected embryos (A). Arrow indicates region of forming mid-hindbrain boundary. (C,D) Ectopic cell death (TUNEL) in response to MoSix3 overlapping with (E) Six3 expression. Broken yellow line outlines forming embryonic axis. (F,G) Lateral views and (H) Rx2 (red) and Vax1 (purple) in situ analysis of (F) GFP control and (G,H) mouse Six3-injected embryos at somitogenesis stage (23); (F-H) transverse section (dorsal is upwards) at the midbrain level of another embryo. Six3 overexpression results in ectopic formation of proximal (Vax1) and distal (Rx2) eye structures in the midbrain (22%; n=37). (I-K) Dorsal views of organogenesis stage embryos (30). (J,K) MoSix3-injected embryos exhibit a small eye (mild) and a cyclopic (intermediate) phenotype. Arrow indicates proximal retinal structures lost, arrowhead indicates forebrain structures lost (K). ey, eye; gt, gut; hb, hindbrain; inj., injected; ot, optic tectum.

View larger version (57K): [in a new window]   Fig. 3. Six3 is involved in proximodistal patterning of the eye. Dorsal views, anterior is towards the left; developmental stages, morpholinos injected and molecular markers used for in situ hybridization are indicated; all Six3 morphants used for analysis exhibit a mild (small eye) phenotype. (B,F,J,N) Broken yellow line outlines optic vesicles. (A,B) Six3 reduction results in loss of Vax1 expression in the optic stalk. (C,D) Loss of forebrain structures and fusion of distal retina structures is visualized by double in situ hybridization with Rx2 (red) and Emx1 (purple). Bar indicates region of remaining Emx1-expressing tissue. (E,F) Retinal Rx3 expression is lost in Six3 morphants. (G-N) In Six3 morphants Six3 is lost and Pax6 is reduced in the retina at six-somite stage (21). mb, midbrain.

View larger version (57K): [in a new window]   Fig. 4. Cooperation of Pax6 and Six3 in eye development. (A-D) Dorsal views of embryos at organogenesis stage, anterior is towards the left; injected morpholinos are indicated. (B) MoPax6 results in development of small eyes compared with control embryos (A). (C,D) Co-interference with Six3 and Pax6 function results in formation of either small (mild) or cyclopic eyes (intermediate). Arrow in D indicates proximal retinal structures lost; arrowhead indicates forebrain lost. (E) MoPax6/MoSix3 co-injection (concentrations in mM) (n=473) shifts the mild (blue) towards the intermediate phenotype (red) when compared with MoPax6/MoGFP (n=50) or MoSix3/MoGFP-injected embryos (n=50). ey, eye; hb, hindbrain; ot, optic tectum.

View larger version (99K): [in a new window]   Fig. 5. Differential effects of MoSix3, MoPax6 or MoSix3/MoPax6 co-injected embryos. Dorsal views of embryos at the six-somite stage, anterior is towards the left; injected morpholinos and molecular markers for in situ hybridization are indicated. (A,B) MoSix3 results in the loss of Pax2 expression. (C,E) Retinal Pax2 is still expressed even in more severely affected MoSix3/MoPax6 double morphants. (F,H,I,K) In MoSix3/MoPax6 double morphants retinal Pax6 but not Six3 expression is reduced at early somitogenesis. (D,G,J) Pax2, Six3 and Pax6 expression are not affected in Pax6 morphants. broken yellow line outlines optic vesicle in B,K. mb, midbrain.