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Isolation and characterization of a downstream target of Pax6 in the mammalian retinal primordium

Gilbert Bernier1, Wolfgang Vukovich1, Lorenz Neidhardt2, Bernhard G. Herrmann2 and Peter Gruss1,*

1 Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, 37077 Göttingen, Germany
2 Max Planck Institute of Immunology, Stübeweg 51, D-79108, Freiburg, Germany



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  		Fig. 1. Necab encodes a phylogenetically conserved mammalian protein. (A) Nucleotide sequence and amino acid translation of mouse Necab cDNA. Necab encodes a 389 amino acid protein (dark blue) with an EF-hand Ca2+-binding domain (in pale blue). A polyadenylation signal is present at position 1944 to 1953 of the nucleotide sequence. (B) Alignment of mouse (Mus musculus; Accession Number AF411253), macacus (Macaca fascicularis; Accession Number AB046031) and human (Homo sapiens) Necab amino acid sequence. Red, amino acid identity in all shown sequences; blue, amino acid identity in two out of three species; green, amino acid similarity; black, divergence. The amino acid stretch in bold of the human sequence represents possible mistakes in the predicted translation product of NECAB cDNA. (C, part I) Immunohistochemistry with Ab1 antibody at 10.5 dpc (coronal sections) showing immunoreactivity in the retinal-pigmented epithelium (rpe) and in the neural retina (nr). No signal is detected in the in the lens (ls) or in the surrounding eye mesenchyme. (C, part II) Immunoreactivity of Ab1 in the Rathke’s pouch (Rp) and in the infundibulum recess (inf). (C, part III) Immunofluorescence at 17.5 dpc (cross sections) with Ab1 antibody. Predominant immunoreactivity is found in the cytoplasm of the ganglion cells (arrowheads). (C, part IV) A Necab/v5 epitope fusion protein is localized to the cytoplasm in COS cells. (D) FISH on metaphase chromosomes. NECAB is localized to human chromosome 16q23 and Necab is localized to mouse chromosome 8E1.

 


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  		Fig. 2. Necab co-expresses with Pax6 in the developing optic vesicles and in the pre-tectum. (A-D) Whole-mount in situ hybridization showing Necab expression in the optic sulcus, optic vesicles (black arrowhead) and pretectum (white arrowhead). (C,D) Frontal and top views of the embryo, respectively, showing the sharp expression domain of Necab in the pretectum. (E) Necab mimics Pax6 expression pattern in the embryonic forebrain. (F) Necab expression is detected in the midbrain at 10.5 dpc, in contrast to Pax6. The arrowhead shows the forebrain/midbrain boundary. (G-J) Necab is expressed in a gradient manner in the optic vesicle (cross sections). Strong expression is observed in the presumptive ciliary margin region (G), similar to Pax6 (H). By contrast, (I) Rx expression is predominant in the retinal plate (RP) and (J) Lhx2 expression is predominant in the inner portion of the optic vesicle (arrowheads). (K) Necab expression is detected in the retinal-pigmented epithelium (rpe) and is predominant in the ciliary margin (cm) of the neural retina (nr), like Pax6. (L). fb, forebrain; hb, hindbrain; lp, lens placodes; ls, lens; mb, midbrain; ov, optic vesicle; prt, pre-tectum; rp, roof plate of the midbrain.

 


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  		Fig. 3. Necab coexpresses with Pax6 in the olfactory placodes, the iris and the ciliary margin of the retina. (A-D) Radioactive in situ hybridization at 10.5 dpc (coronal sections) showing co-expression of Necab and Pax6 in the retinal-pigmented epithelium (rpe), the neural retina (nr), the olfactory placodes (op), the forebrain (fb) and the pre-optic area (poa). Necab is also expressed in the dorsal portion of the midbrain (A, arrows). Arrowhead in B indicates the lens pit. (E,F) Necab is expressed in the dorsal thalamus (dt), the roof plate of the midbrain (rp), and at low level in the hypothalamus (ht), (coronal sections, 13.5 dpc). (G,H) Necab and Pax6 expression is detected in the distal portion of the retina (cm) and in the future iris (white arrowhead). The black arrowhead indicates the cornea. (I) Necab is expressed in the ciliary margin (cm) and iris (17.5 dpc), and at low level in the ganglion cells layer (gc). (J,K) Necab and Pax6 are co-expressed in the ciliary margin and in the iris (white arrowhead) (17.5 dpc). ls, lens; vt, ventral thalamus.

 


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  		Fig. 4. Necab is downstream of Pax6. (A) X-gal staining showing Pax6 expression in the spinal cord, forebrain and optic vesicles. Immunofluorescence showing ß-galactosidase-positive cells in the optic vesicle of a Pax6 mutant embryo (Inset). (B-F) In situ hybridization showing that, in contrast to Otx2 (B), Six3 (C), Lhx2 (D) and Rx (E), Necab expression is absent in the optic vesicle and in the pretectum of Pax6 mutant embryos (arrowhead, F). Wild type, +/+; heterozygous Pax6 mutant, +/-; homozygous Pax6 mutant, -/-.

 


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  		Fig. 5. Pax6 can induce ectopic expression of Necab. (A) Schematized view of the electroporation procedure. Mouse embryos are microinjected with a needle containing the DNA solution (in blue) and electroporated. (B,C) Double whole-mount in situ hybridization (8.5 dpc), 6 hours after electroporation with a Pax6 expression vector. (B) Ectopic expression of Necab is detected in a subset of cells (blue arrowhead). Endogenous expression is detected in the pretectum and optic vesicle (white arrowheads). (C) The expression domain of the Pax6 transgene (red arrowhead) is broader but overlaps ectopic Necab-positive cells. (D-F) Double in situ hybridization (8.5 dpc) on midbrain explants. (D,E) Twenty four hours after electroporation with a Pax6 expression vector, cells expressing the transgene (in red) are also expressing Necab (in blue). (E) Control explants (no electroporation) cultured for 24 hours and hybridized with both probes. (G,H) Double in situ hybridization (8.5 dpc), 6 hours after electroporation with a Six3 expression vector. Transgene expression is present (red arrowhead) but ectopic Necab-expressing cells are not detected. (I) Cross section at the hindbrain level of the embryo in G, showing that the transgenic cells do not expressed Necab. (J,K) Six hours after the electroporation of a Necab expression vector, ectopic expression of Chx10 is detected in the midbrain region (J) where transgenic cells are present (K). Red arrowhead, transgene expression; blue arrowhead, ectopic expression of the target gene; white arrowhead, endogenous expression.

 

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