The homeobox gene, Six3, is a vertebrate homologue of Drosophila sine oculis, which is essential for development of the visual system (Cheyette et al., 1994). In early mouse embryos, Six3 is expressed in the anterior-most neural plate, and subsequently also in the developing eye (Oliver et al., 1995). We have found that a 9 kb genomic DNA fragment of the mouse Six3 (Fig. 1a) contains regulatory elements that can drive transgene expression in an eye-and forebrain-specific manner (OL and GCO, unpublished results). Using this fragment, we have generated the transgene, designated Six3A1A2-Cre, in which the cDNA encoding Cre recombinase with the nuclear localization signal of the SV40-T gene (Kalderon et al., 1984) is inserted into the first coding exon (Fig. 1b). Purified transgene fragment is injected into pronuclei of fertilized eggs (C57BL/6 DBA2 F2). We have obtained six transgenic founder mice (Cre# 22, 41, 46, 51, 56, and 69), and they have been backcrossed to C57BL/6 animals to be maintained as lines. To test for Cre activity in vivo, F1 or F2 hemizygous Cre transgenic females are crossed with males that have the ROSA26 reporter (R26R) allele (Soriano, 1999). Embryos are recovered at day 8.5–14.5 (E8. 5–14.5), and-galactosidase (-gal) activity resulting from Cre-mediated recombination of the R26R locus is monitored by X-Gal staining. Four out of six lines exhibit retina-and ventral forebrainspecific Cre activity starting at E9. 0–9.5. Figure 2a shows a typical X-Gal staining pattern in the eye and ventral forebrain in an E9. 5 (27-somite stage) embryo trans-hemizygous for Cre# 51 and R26R. Sections of the eye of such stained embryos reveal a high frequency of recombination in the retina and the ventral aspect of the optic stalk by this stage (Fig. 2b). In lines Cre# 46, 51, and 69,-gal activity is detected as early as E9. 0 (20-somite stage) in these regions (data not shown), and by E12. 5, the entire retina becomes positive for-gal activity (Fig. 2c). Staining is also found in the retinal pigmented epithelium, optic stalk, and optic nerve (Fig. 2c). In line# 22, however, the onset of-gal activity in the retina is slightly later (E9. 5) compared to the other three lines, and it is not until E14. 5 that the entire retina becomes positive for-gal activity (not shown).

Recombination in the ventral forebrain is detected from E9. 0 in all four lines.-gal positive cells appear less frequent in this region compared to those in the retina at E9. 5, and the number gradually increases as development proceeds (data not shown). By E14. 5,-gal activity is widely detected in the ventral forebrain, such as the basal ganglia, medial amygdaloid area, hypothalamus