rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina

Abstract

Development of the vertebrate eye has been found to require the activity of several genes encoding homeodomain proteins (Freund, C., Horsford, D. J. & McInnes, R. R. (1996) Hum. Mol. Genet. 5, 1471-1488). Some of these genes, or portions thereof, are highly conserved across phyla. In this paper, we report the identification of a novel homeobox gene, rax (retina and anterior neural fold homeobox), whose expression pattern suggests an important role in eye development. The predicted amino acid sequence of Rax comprises a protein with a paired-type homeobox, as well as the octapeptide that is found in many paired-type homeobox genes. In addition, in the C terminus of Rax, we found a 15-aa domain that we have named the OAR domain. This domain is also found in several other homeobox genes. In the early mouse embryo, rax is expressed in the anterior neural fold, including areas that will give rise to the ventral forebrain and optic vesicles. Once the optic vesicles form, rax expression is restricted to the ventral diencephalon and the optic vesicles. At later stages, rax expression is found only in the developing retina. After birth, the expression of rax is restricted to the zone of proliferating cells within the retina, and expression gradually decreases as proliferation declines. These findings suggest that rax is one of the molecules that define the eye field during early development and that it has a role in the proliferation and/or differentiation of retinal cells.

Figure 1

rax nucleotide and amino acid sequences. Boxed amino acids are the homeodomain sequence. The dashed box shows the octapeptide sequence. Double underline indicates the OAR domain. Possible initiation methionines are in boldface type.

Figure 2

(A) Alignment of homeodomain sequences for the paired-type homeodomain-containing proteins. Only amino acid residues that differ from those of the Rax homeodomain are shown. (B) Sequence alignment of OAR domain sequences. Conserved identical amino acid residues are shown with a dark shadow. Conservative changes of amino acid residues are shown with a light shadow. Sequences of homeodomains and OAR domains are taken from the following references: aristaless (33), chx10 (20), pax3 (35), pax6 (10), pax7 (36), alx3 (37), phox2 (38), s8 (39), cart1 (40), rk2 (41), paired (34), and otp (42).

Figure 3

Chromosomal location of the rax gene on mouse chromosome 18. (Left) Recombination fractions for adjacent loci: the first fraction represents data from the M. spretus crosses, and the second fraction is from the M. m. musculus crosses. In parentheses are recombinational distances and standard errors calculated according to Green (29). (Right) The positions of loci in human chromosomes, where known.

Figure 4

Whole-mount in situ hybridization of E7.5 (A), E8.5 (B), E9.5 (C and D), E10.5 (E and F), and E11.5 mouse embryos (G and H). (Left) Anterior side of the embryos. (A) rax expression is detected in the cephalic head fold. (B) rax expression is at a high level in the prospective forebrain. (C) The embryo has finished turning and the optic vesicles have been formed. The optic vesicles exhibit a high level of expression of rax. (D) A ventral view of the embryo shown in C. rax expression is detected in the optic vesicles, optic stalk, and ventral diencephalon. (E–H) Lateral view of the embryos at E10.5 (E and F) and E11.5 (G and H). rax is expressed in the optic cup. F and H represent a higher magnification of E and G, respectively. Note that the optic fissures are negative for rax expression.

Figure 5

Comparison of the expression patterns of rax, six3, otx2, and pax6 during optic vesicle formation. (A, B, D, and E) Dorsal views of E8.5 mouse embryos. (C) A dorsal view of an E8.5 embryo after turning. (F–I) Side views of E9.0 embryos. Hybridization probes are as follows: (A and F) rax, (B, C, and G) six3, (D and H) otx2, (E and I) pax6. Arrowheads indicate the level where the optic vesicles begin to evaginate. Note that only rax expression is specific to the optic vesicles and adjacent ventral diencephalon.

Figure 6

rax expression during development of the mouse retina. (A) A coronal section of the forebrain of an E9.5 mouse embryo. The rax hybridization signal was detected in the optic vesicles and the ventral diencephalon. (B) A section through an E11.5 eye. (C) A section through an E18.5 eye. rax expression is not observed in the ganglion cell layer. (D–F) Cross-sections of a P0, P6, and adult mouse retina. Note the stronger signal in the peripheral retina, which lags in development behind that of the central retina. (G–I) Higher magnification of the cross-section of the retina shown in D–F. At P0 and P6, the outer nuclear layer and the inner nuclear layer are developing. Progenitor cells in the VZ are intermixed with these layers. ov, optic vesicle; gcl, ganglion cell layer; l, lens; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; opl, outer plexiform layer; os, outer segment; vz, ventricular zone.

Figure 7

Northern hybridization analysis of rax expression in adult mouse organs. (Upper) Hybridization signals obtained with the mouse rax cDNA. (Lower) Ethidium bromide staining of the RNA. The rax transcripts are ≈4.0 kb and 1.8 kb in the P0-P3 retina, which is used as a control for hybridization.