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Fig. 6. Regulation of early eye field formation by Hip and Shh. (A) (a, g) Frontal views of embryos at NF stage 14, injected side to the right. (f, l) Lateral views of e and k. (a) Increased number of retinal progenitors upon mHip1 overexpression (750 pg; 71%, n = 46). (g) Reduction of Xrx1 expression after injection of Shh (500 pg; 50%, n = 32). (b, h) While Shh overexpression results in a slight reduction of Pax6 expression in the anterior neural plate and Pitx1 expression in the early lens field (45%, n = 11), both genes are up-regulated and expanded laterally after mHip1 injection (67%, n = 9). Microinjection of mHip1 mRNA results in strongly expanded domains of Six3 and Otx2 (c, 60%, n = 10; d, 100%, n = 8), but are almost normal upon Shh overexpression (i, 23%, n = 13; j, 67%, n = 12). (e, f) Overexpression of mHip1 results in reduction and lateral shift of Dlx3 (black arrows, 43%, n = 14). (k, l) Expression of Dlx3 in ectoderm surrounding the neural plate is increased after Shh injection (black arrows, 36%, n = 14). (B) (a) Frontal views of embryos at NF stage 13, injected side to the right. (a) Strong activation of Six3 upon injection of dnWnt-8 mRNA (63%, n = 11), while Dlx3 expression is reduced (b, 58%, n = 12). XFD overexpression did not alter Six3 (c, 95%, n = 9) or Dlx3 (d, 100%, n = 8) expression significantly. LacZ mRNA was co-injected and visualized by red- gal stain. (C) (a) BrdU-proliferation assay and (d) phospho-histone H3-proliferation assay. Frontal views of embryos at NF stages 14 and 15, respectively. mHip1 and Shh mRNA was injected into one cell of a two-cell stage embryo, injected side to the right. (b, c) Rx1 expression (red) was monitored to visualize mHip1 and Shh activity (indicated by dashed line). (b, e) The number of BrdU- and p-H3-positive cells is slightly enhanced by Shh and reduced upon mHip1 overexpression (c, f) versus control embryos (a, d). (g) Frontal views of tailbud embryos at NF stage 33 after TUNEL staining. (h) Shh overexpression reduces cell death, whereas the number of apoptotic cells is increased upon mHip1 injection (i). LacZ mRNA was co-injected as a lineage tracer (light blue). (jl) Mitotic arrest assay. (j) Control embryo, showing that normal Rx1 expression is maintained after HUA treatment. (i) mHip1 injection still expands the retinal territory (71%, n = 34), whereas in Shh injected embryos Rx1 expression is reduced (k). |
