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Medaka eyeless is the key factor linking retinal determination and eye growth

Felix Loosli1, Sylke Winkler1, Carola Burgtorf1, Elisa Wurmbach2,*, Wilhelm Ansorge2, Thorsten Henrich1,{ddagger}, Clemens Grabher1, Detlev Arendt1, Matthias Carl1, Annette Krone1, Erika Grzebisz1 and Joachim Wittbrodt1,§

1 European Molecular Biology Laboratory, Developmental Biology Programme, Meyerhofstr. 1, 69117 Heidelberg, Germany
2 European Molecular Biology Laboratory, Biochemical Instrumentation Programme, Meyerhofstr. 1, 69117 Heidelberg, Germany
* Present address: Mount Sinai School of Medicine, New York, NY 10029, USA
{ddagger} Present address: Kondoh differentiation signalling project, Kyoto, Japan



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  		Fig. 1. Rx3 expression in wild-type and el-mutant embryos. Dorsal views of whole-mount in situ hybridisation of late gastrula (stage 16; A,B) and four-somite stage (stage 20; C,D) wild type (A,C) and el-mutant embryos kept at restrictive temperature (B,D). Anterior is towards the left. Neural axis is indicated by a broken yellow line (A,B). (A) Rx3 expression in anterior neural plate. (C) Rx3 expression in hypothalamus (arrow) and optic cup (arrowhead). (B,D). Note complete loss of Rx3 expression in mutant embryos kept under restrictive conditions. (F) Under permissive conditions, Rx3 is weakly expressed in forebrain of el-mutant embryos at the two-somite stage (stage 19). Note reduced size of evaginating optic vesicles (arrowhead) at site of reduced Rx3 expression compared with wild-type embryo of same stage (arrowhead in E). (G) Primers spanning entire open reading frame detect the wild-type transcript by RT-PCR in wild-type (lane 1) and el-mutant embryos (lane 2) under permissive conditions, while no transcript is detected in mutants under restrictive conditions (lane 3). M, 100 bp ladder. Abbreviations: perm., permissive temperature; restr., restrictive temperature.

 


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  		Fig. 2. Molecular analysis of the genomic Rx3 region. (A) CAPS analysis of mutant and wild-type genomic DNA. A 190 bp fragment in HaeIII digested genomic PCR fragment is diagnostic for the mutant Cab allele (el/el and el/+ lanes); the additional restriction site in wild-type Kaga results in 100 bp and 90 bp fragments (arrowhead, el/+ and +/+ lanes). (B) Genomic Southern blot hybridisation using a 600 bp HindIII cDNA fragment comprising exon 1 and 2 (BamHI digest) and a 3 kb genomic DNA fragment (broken line in D; XbaI digest) as probes. In DNA of homozygous mutant embryos (el/el) a larger fragment is detected than in the wild type (+/+), indicating an insertion larger than 13 kb in the mutant el locus. In DNA of heterozygotes (el/+), both fragments are detected. (C) Genomic Southern blot hybridisation with a 4 kb insertion fragment reveals multiple copies in the genome, indicative of middle repetitive DNA. (D) Map of Rx3 region indicating relative distance of B and el on linkage group 12 (LG 12). Red boxes represent three exons on Rx3 cosmid; position of the insertion in intron 2 is shown. Regions comprised in different rescue plasmids and position of the frameshift (fs) are indicated. (E) Predicted amino acid sequence of the Rx3 protein. Splice sites are indicated (arrowheads). Conserved octapeptide (yellow), homeodomain (green) and C-terminal region (red) are highlighted. Abbreviations: B, BamHI; Be, BspEI; S, SnaBI; X, XbaI.

 


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  		Fig. 3. Phenotypic rescue of el-mutant embryos. Uninjected (A,B) and injected (C,D) wild-type (B) and mutant embryos (A,C,D) raised for 8 days at restrictive temperature. Dorsal views, anterior is towards the top. Note dark pigmented melanophores in wild-type embryo (arrowhead in B), lacking in mutants (A,C,D). (A) No eyes form in mutants. (C,D) Rescued eye formation in mutant embryos injected with Rx3 plasmid. (C) Note complete rescue of both eyes. (E) Table displaying results of different DNA and control injections. N, number of injected embryos; el (b/b) homozygous mutant embryos as judged by absence of dark melanophores. No rescue is observed in control injected embryos.

 


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  		Fig. 4. Restored gene expression in rescued mutant embryos. (A-F) Rx3 expression at the four-somite stage in mutant (A-C,E) and wild-type (D,F) embryos, dorsal views (A-D,F), injected with Rx3 plasmid (A,B), Rx3{Delta}HB plasmid (C,D) and controls (E,F); lateral view (E). Anterior is towards the left. (A,B) Optic vesicle evagination correlates with level of restored Rx3 expression (compare arrowheads in A,B). (C) Restored expression of nonfunctional Rx3 protein does not rescue. (A-D) Note specific Rx3 expression in mutant (A-C) and wild-type (D) embryos. Compare the homogenous wild-type Rx3 expression in control injected (F) and clonal expression in Rx3 plasmid injected mutant (A-C) and wild type (D) embryos. (E) Control injected mutant embryo lacks Rx3 expression. (G-L) Rx3 plasmid (G,J) and control injected (H,I,K,L) 12-somite mutant (G,I,J,L) and wild-type (H,K) embryos. Tbx2 and Tbx3 expression in the retina is rescued in Rx3 plasmid-injected embryos (compare arrowheads in G-I for Tbx2, and J-L for Tbx3). Note that expression of Tbx2 and Tbx3 in the hypothalamus (arrows in G-I and J-L, respectively) and Tbx2 expression in the otic vesicle (compare G-I) is not affected by the el mutation. (M-P) Transversal section of 22-somite (M,N) and 35-somite (O,P) uninjected wild-type (M,O) and Rx3 plasmid-injected mutant embryos (N,P). Dorsal is towards the top. (M,N) Vsx2 expression in wild-type (M) and rescued mutant embryo (N) in the ventral retina. (O,P) Vsx1 expression in inner nuclear layer of the wild type retina (O) is rescued in injected mutant embryo (P). Abbreviation: ov, otic vesicle.

 


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  		Fig. 5. Rx3 is not required for retina determination. Dorsal views of two-somite stage (A-F) and six-somite stage el-mutant embryos (G,H). Anterior is towards the left. (A,E,G) Mouse Six3 RNA injected, (C) medaka Six3 RNA injected, (B,D,F,H) control injected el-mutant embryos raised at restrictive temperature. (A,B) Six3 overexpression does not result in Rx3 expression in el-mutant embryos. (B,C) Ectopic Pax6 expression (arrowhead) in the midbrain in response to Six3 overexpression. (E) Six3 overexpression results in ectopic Six3 expression in the presumptive midbrain (arrowhead) (G) Overexpression of Six3 results in expanded and ectopic Rx2 expression (arrowhead) in forebrain and presumptive midbrain, respectively. Abbreviations: fb, forebrain; hb, hindbrain; mb, midbrain.

 


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  		Fig. 6. Regulatory interactions of Six3 and Rx3. Dorsal views of late gastrula (stage 16, A-D), two-somite stage (stage 19, E and F) and six-somite stage embryos (stage 21, G-L). Anterior is towards the left. (A-C) The expression of Six3 (A), Rx3 (B) and Pax6 (C) partially overlap in presumptive forebrain. Anterior end of neural axis indicated by a broken line. (D,E) Overexpression of Six3 results in expanded Rx3 expression (arrowhead) and ectopic expression in the presumptive midbrain (arrow) at early neurula stage (D) and two-somite stage (E). (E) Note expanded expression in enlarged optic vesicle (arrow). (F) Control injected embryo showing wild-type expression. (G-I) Rx3-injected embryos with enlarged optic vesicles (arrowhead) show expanded Six3 (G), Pax6 (H) and Rx2 (I) expression. (J-L) Control injected embryos at same stage. Note that anterior and posterior boundaries of respective expression domains are not shifted in response to Rx3 overexpression (compare G with J,H with K and I with L). Insets in I and L show horizontal sections at comparable level. Note the increased cell number in the Rx3-injected eye (I).

 


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  		Fig. 7. Genetic hierarchy underlying early retina development. X, Y and Z represent factors that pattern the anterior neuroectoderm, leading to the overlapping expression of Six3 and Pax6 in the retina anlage, which results in the determination of retinal fate. The crossregulatory interaction and the feedback loops of Six3 and Pax6 (arrows) result in the maintenance of retinal fate. Rx3 expression is regulated by these genes, but may also receive input of upstream factors X, Y and Z. Rx3 activity is required for morphogenesis (optic vesicle evagination) and organ size regulation (proliferation in optic vesicle). The retina specific expression of Rx2 does not depend on Rx3 activity. Genes involved in subsequent differentiation steps require preceding Rx3 activity (Tbx2/3, Vsx1/2).

 

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© The Company of Biologists Ltd 2001