Portal de Periódicos da CAPES

Six3 activation of Pax6 expression is essential for mammalian lens induction and specification

2006; Springer Nature; Volume: 25; Issue: 22 Linguagem: Inglês

10.1038/sj.emboj.7601398

1460-2075

Wei Liu, Oleg Lagutin, Michael Mende, Andrea Streit, Guillermo Oliver,

Developmental Biology and Gene Regulation

Article26 October 2006free access Six3 activation of Pax6 expression is essential for mammalian lens induction and specification Wei Liu Wei Liu Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Oleg V Lagutin Oleg V Lagutin Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michael Mende Michael Mende Department of Craniofacial Development, King's College London, London, UK Search for more papers by this author Andrea Streit Andrea Streit Department of Craniofacial Development, King's College London, London, UK Search for more papers by this author Guillermo Oliver Corresponding Author Guillermo Oliver Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Wei Liu Wei Liu Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Oleg V Lagutin Oleg V Lagutin Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michael Mende Michael Mende Department of Craniofacial Development, King's College London, London, UK Search for more papers by this author Andrea Streit Andrea Streit Department of Craniofacial Development, King's College London, London, UK Search for more papers by this author Guillermo Oliver Corresponding Author Guillermo Oliver Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Author Information Wei Liu1, Oleg V Lagutin1, Michael Mende2, Andrea Streit2 and Guillermo Oliver 1 1Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA 2Department of Craniofacial Development, King's College London, London, UK *Corresponding author. Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA. Tel.: +1 901 495 2697; Fax: +1 901 526 2907; E-mail: [email protected] The EMBO Journal (2006)25:5383-5395https://doi.org/10.1038/sj.emboj.7601398 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction. Introduction In vertebrates, the lens originates from the presumptive lens ectoderm (PLE). The lens placode, a thickened region of the head surface ectoderm (SE), is the first morphologic sign of lens development. Interactions between the lens placode and the optic vesicle lead to the formation of the lens pits and neuroretina. Lens induction begins in the head SE before contact between the PLE and optic vesicle is established (Grainger et al, 1992, 1997; Sullivan et al, 2004). The homeobox gene Pax6 is an evolutionarily conserved key regulator of metazoan eye development (Hill et al, 1991; Jordan et al, 1992; Glaser et al, 1992; Quiring et al, 1994; Ashery-Padan et al, 2000), and Pax6 heterozygosity results in ocular abnormalities such as aniridia in humans (Glaser et al, 1994) and small eye in rodents (Hogan et al, 1986; Hill et al, 1991; Matsuo et al, 1993). Furthermore, in Pax6−/− mice, the lens and other eye structures do not form (Hill et al, 1991; Grindley et al, 1995). Conditional deletion of Pax6 in the mouse lens prevents lens placode formation (Ashery-Padan et al, 2000). Lang (2004) has proposed that the preplacodal phase of Pax6 expression regulates the later placodal phase of expression. In this context, BMP and FGF signaling act upstream of the Pax6 pathway controlling lens placode formation (Lang, 2004). Approximately 40 conserved, noncoding sequences potentially involved in Pax6 regulation have been identified in the Pax6 locus (see trafac.cchmc.org), and two Pax6 enhancers, ectoderm enhancer (EE) (Williams et al, 1998; Kammandel et al, 1999; Xu et al, 1999; Dimanlig et al, 2001) and SIMO enhancer (Kleinjan et al, 2001), are important for lens lineage expression. However, the upstream regulators of the preplacodal phase of Pax6 have remained elusive. Meis family proteins regulate Pax6 activity in the PLE (Zhang et al, 2002); however, Meis1-deficient embryos have partially duplicated retinae and small lenses, which suggests that either Meis2 compensates for Meis1 or other unidentified factors regulate Pax6 in the PLE (Hisa et al, 2004). Heterozygous mutations in the transcription factor Sox2 that produce hypomorphic conditions occur in about 10% of humans with anophthalmia or severe microphthalmia (Fantes et al, 2003; Hagstrom et al, 2005; Ragge et al, 2005; Zenteno et al, 2005). Sox2 in known to regulate δ-crystallin expression in the chick in a complex with Pax6 (Kamachi et al, 2001). Sox2-null mice die at preimplantation (Avilion et al, 2003), thereby preventing analysis of this gene's role(s) in eye development. Sox2 expression is absent in mutant mouse strains with severe lens defects such as Bmp4, Pax6sey/sey and Bmp7 (Furuta and Hogan, 1998; Wawersik et al, 1999). Sox2 expression precedes lens placode formation (Furuta and Hogan, 1998) and its upregulation in the PLE is one of the earliest indicators of lens specification and is dependent on Pax6 (Furuta and Hogan, 1998; Zygar et al, 1998). Upregulation of Sox2 was not detected in the SE of Pax6Sey-1Neu-homozygous embryos in which lens induction is defective (Furuta and Hogan, 1998). However, the early unaffected phase of Pax6 expression in the SE of Pax6-Le conditional mutant embryos was sufficient to upregulate Sox2 expression in this tissue (Ashery-Padan et al, 2000), a finding that indicates that upregulation of Sox2 in the PLE is dependent on early, but not late Pax6 activity. Six3 protein expression was reported to start at around E9.5 in the vertebrate lens placode (Oliver et al, 1995), and forced Six3 expression in fish and mouse embryos promotes ectopic lens (Oliver et al, 1996) and neural retina formation (Loosli et al, 1999; Lagutin et al, 2001). Until recently, investigating the in vivo role of Six3 in eye development was not possible, because the prosencephalon, the brain region from which eyes form, is severely truncated in Six3−/− mice (Lagutin et al, 2003). However, by using a Cre/loxP approach to inactivate Six3 in the developing eye, we can now address this question. Results Cre/loxP-mediated removal of Six3 activity from the PLE To address the in vivo role of Six3 during lens morphogenesis, we generated the floxed Six3 mouse line Six3f/w (nomenclature and details in Supplementary Figure S1). During the initial crosses of Six3f/w mice with the Le-Cre mouse strain (Ashery-Padan et al, 2000), the transgenic Cre strain was occasionally germline active. This allowed us to generate the Six3Δ allele in which the floxed sequence encoding the Six domain and homeodomain of Six3 was permanently deleted (Supplementary Figure S1). The generation of the Six3Δ/Δ mice confirmed that the Six3Δ allele was a true null allele; the prosencephalon truncation in Six3Δ/Δ mice was indistinguishable from that previously reported in Six3−/− mice (Lagutin et al, 2003). Furthermore, antibody staining of the generated Six3-mutant lens failed to detect any signal (see below) when we used an anti-Six3 antibody (our unpublished data) that recognizes a Six3 epitope downstream of the floxed DNA sequence. Six3Δ/w;Le-Cre mice were generated by crossing Six3Δ/w mice with Le-Cre mice in which Cre activity in the PLE was reported to start at around E9.0 (Kammandel et al, 1999; Ashery-Padan et al, 2000). Six3f/f mice were generated by intercrossing Six3f/w mice. Finally, Six3f/Δ;Le-Cre mice were obtained by crossing Six3f/f females with Six3Δ/w;Le-Cre males. Six3f/w embryos, which were indistinguishable from wild-type embryos, were used as controls. Six3 mediates lens morphogenesis Gross morphological analysis of the generated Six3f/Δ;Le-Cre mice revealed several lens phenotypes, including drastically reduced lens size, cataracts, or absence of the lens (Figure 1A–D, A′–D′). These defects were even more obvious in the analysis of lens sections. Three major phenotypes were observed at any particular stage: mild (type I), moderate (type II), and severe (type III). A summary of this analysis is included in Supplementary Table 1. Figure 1.Conditional deletion of Six3 in the PLE causes severe lens phenotypes. Compared to age-matched control lenses (A–D), Six3f/Δ;Le-Cre mutant eyes showed a range of lens defects at representative stages. (A′) At E12.5, the mutant eye appeared smaller, and the shape was abnormal. The shape of the retinal-pigmented epithelium (arrowhead) was defective. (B′) At E14.5, defects in the shape of the mutant eyes were consistently identified, and the lens (arrowhead) was extremely reduced. (C′) Three-month-old isolated mutant eyes exhibited obvious retinal defects such as irregular shape (arrow). The isolated lens was very small and degenerated and appeared to have cataracts (arrowhead). (D′) At 6 months, the eye lids of Six3f/Δ;Le-Cre mutant mice were closed (arrowhead), suggestive of anophthalmia or microphthalmia. (E–P) H & E–stained sections of control and Six3f/Δ;Le-Cre-mutant lenses. At E10.5, some mutants exhibited a smaller abnormal lens pit (arrows in F, G). (H) No lens pit (arrow) was present in those with the most severe phenotype, and the retina appeared to have been duplicated (arrowhead). At E12.5, some type I mutants exhibited slightly reduced lens vesicle (arrow in J), and some type II mutants showed greatly reduced ones (K). No lens vesicle was present in type III mutants (arrow in L). Similar phenotypic variations were seen at E14.5. The lens was smaller (N) or very reduced and abnormal (O, higher magnification in P). The lens stalk was frequently observed (arrow in N). Download figure Download PowerPoint Defects in lens development were evident as early as E10.5. Reduction in the size of the invaginating lens pit was observed in mildly (not shown) and moderately affected mutant embryos (Figure 1F and G). Embryos with the severe (type III) lens phenotype showed minimal thickening and no invagination of the PLE (Figure 1H). The shape of the optic vesicle was also defective (Figure 1H), although Six3 was not removed from it (Figure 3B′ and E′). Conditional deletion of Pax6 from the PLE also resulted in defective invagination of the optic vesicle (Ashery-Padan et al, 2000). Variability in the severity of lens defects was also observed at later stages. At E12.5, the lens vesicle was small but relatively normal in mildly affected mutant embryos (Figure 1J), smaller and abnormal in others (Figure 1K), and totally absent in type III embryos (neuroretina was also deformed; Figure 1L). At E14.5, some embryos had an abnormally persistent lens stalk and disorganized lens fibers (Figure 1N), and others had a very small and abnormal lens (Figure 1O and P). Thus, removal of Six3 from the PLE during lens induction disrupts lens formation. The time and extent of Le-Cre-mediated Six3 excision from the PLE is variable As mentioned before, Le-Cre activity in the PLE was reported to start around E9.0. (Kammandel et al, 1999; Ashery-Padan et al, 2000). To determine whether the variability in the severity of the lens phenotype reflects differences in the timing and extent of Six3 excision from the PLE, we characterized the temporal and spatial activity of Le-Cre by using the R26R strain (Soriano, 1999). We found that the time and extent of Le-Cre activity was variable (Supplementary Figure S2). At around the 22-somite stage, the most common and rather specific Cre activity was detected in the lens SE (Supplementary Figure S2A and B). In addition, a less common and more variable phase of Le-Cre activity was also identified in the putative lens SE as early as the 7- to 11-somite stage (Supplementary Figure S2C–I); variations in the time and extent of Le-Cre were observed between the left and right eyes of individual embryos (Supplementary Figure S2C and D). X-gal+ cells were also found in mesenchymal and neural cells (Supplementary Figure S2G–I), and in a few cases, X-gal-expressing cells were ubiquitously distributed along most of the embryo (Supplementary Figure S2F). Consistent with these results, we also found variability in the time and extent of Cre protein expression in the PLE; extensive Cre expression was seen in the PLE of a 16-somite-stage embryo (Supplementary Figure S2J); in contrast, fewer positive cells were seen in the same region of a somite-matched littermate (Supplementary Figure S2K). According to these results, the variability in the time and extent of Six3 removal from the PLE (compare Figure 3B′ with Supplementary Figure S3D and G) and, therefore, the severity of the Six3 lens phenotype are probably direct consequences of the variable onset of Le-Cre activity. In some E9.5 Six3-mutant embryos, high levels of Six3 remained in the PLE (Supplementary Figure S3D and G); therefore, the levels of Pax6 and Sox2 expressed in the PLE were normal or only mildly reduced (Supplementary Figure S3E and F). These differences could account for the milder (types I and II) phenotypes observed in some Six3-mutant embryos at later stages. In this experimental setting, Six3 deletion from the PLE probably ranged from about the 7-somite stage to E9.5; from E9.0 to E9.5 for most of the characterized less severe lens mutant embryos; and starting from the 7-somite stage onward for the reduced percentage of embryos that exhibited the most severe lens phenotype. Lens differentiation is not affected in mildly or moderately affected Six3-mutant lenses Six3 mutants with mild lens phenotypes (types I and II) have lens pits and lens vesicles, indicating that lens placode formation was not severely affected. This conclusion was supported by the normal expression of the early lens placodal markers Pax6, Sox2, Mab21l1, sFrp2, and FoxE3 at E10.5 (data not shown), and FoxE3 (Figure 2A′) and Prox1 (Figure 2B′) at E12.5. Lens differentiation also appeared unaffected, as shown by the normal expression of β-crystallin (Figure 2C′). However, an increase in cell death was observed in the lens pit at E10.5 (see Figure 2D′), and although the percentage of BrdU+ cells was unaffected at this stage (Figure 2E′), at E12.5 proliferating cells were abnormally located in the posterior lens compartment (Figure 2F′). Therefore, in moderately affected Six3-mutant lenses, cells in the posterior margin do not exit the cell cycle, and lens polarity is probably not established, as also indicated by the ectopic expression of FoxE3 in that region (Figure 2A′). Figure 2.Lens specification and differentiation is not altered in the moderately affected Six3f/Δ;Le-Cre mutants. Expression of FoxE3 (A′), Prox1 (B′), and β-crystallin (C′) in the moderately affected E12.5 lenses indicated that placode formation and lens differentiation had occurred in some Six3f/Δ;Le-Cre embryos. Nevertheless, the polarity of the lens vesicle was affected; persistent abnormal FoxE3 expression was detected in the posterior part of the lens vesicle (A′, arrow). (D′) Increased apoptosis was detected in E10.5 Six3-mutant lens pit (arrowhead in rectangle; 3±1% for the control, 25±5% for the mutant, n=3). (E–E′) The percentage of BrdU+ cells was normal in E10.5 Six3-mutant lens pit (arrowhead in rectangle; 42±0.6% for the control, 41±1% for the mutant, n=3); however, ectopic BrdU+ cells were found in the posterior part of the lens vesicle at E12.5 (arrowheads in F′). Download figure Download PowerPoint Figure 3.Lens induction and specification are defective in the severely affected Six3f/Δ;Le-Cre-mutant lens. Cre activity was restricted to the PLE in E9.5 (A) and E10.5 (A′) Six3f/Δ;Le-Cre-mutant embryos. (B′) Six3 expression was deleted from the mutant PLE (arrow) but was unaffected in the developing optic vesicle (arrowhead). (C′) Pax6 expression in the PLE was drastically reduced (arrow), and that of Sox2 was absent (arrow in D′); however, Pax6 and Sox2 expression in the optic vesicle remained normal (arrowheads in C′, D′). (E′–K′) Lens induction and specification was arrested, as indicated by the lack of thickening or invagination of the PLE and the absence of Sox2 expression. In E10.5 Six3f/Δ;Le-Cre mutant embryos, Six3 expression in the PLE was deleted (arrows in E′) but not that in the neural retina lineage. In this embryo, the optic vesicle appeared to be duplicated. (F′) Pax6 expression was downregulated in the PLE (area between arrowheads), and that of Sox2 (G′, region between the two arrows) was absent. Expression of lens differentiation markers Prox1 (H′), sFrp2 (I′), and FoxE3 (J′) was not detected in the mutant tissue, where the lens pit should have formed. (K′) Expression of Meis1 remained unchanged. Download figure Download PowerPoint Lens induction/specification is defective in the severely affected Six3-mutant lens Our studies focused on the severe lens phenotype (type III), which most likely resulted from early (7-somite stage onwards), extensive removal of Six3 activity from the PLE. Le-Cre activity was already abundant in the PLE of Six3-mutant embryos at E9.5 (Figure 3A) and started decreasing at E10.5 (Figure 3A′). Lens specification normally occurs at around E9.0–E9.5. In severely affected embryos, Six3 expression was already removed from the mutant PLE at E9.5 (Figure 3B′); Six3 expression in the neural retina remained unaffected. Pax6 expression was drastically reduced (Figure 3C′), and Sox2 expression was absent in the mutant PLE (Figure 3D′). The few Pax6-expressing cells remaining in some mutant PLE were most likely remnants of Pax6 that was expressed before Cre-mediated deletion of Six3. Consistent with these results, Six3 expression was not detected in the mutant PLE at E10.5 (Figure 3E′) but was normal in the neural retina, and no lens-like structures were present, as indicated by the absence of thickening and invagination of the SE. In adjacent sections, Pax6 expression was barely detectable (Figure 3F′) in the mutant SE, and Sox2 was absent (Figure 3G′). Consequently, expression of downstream markers of the invaginating lens pit such as Prox1 (Figure 3H′), sFrp2 (Figure 3I′), and FoxE3 (Figure 3J′) was also undetected in the Six3-mutant lens. Expression of Meis1 (Figure 3K′) and Meis2 (data not shown), which directly regulate Pax6 expression in the lens placode (Zhang et al, 2002), appeared unaffected. Six3 is a direct transcriptional regulator of Wnt1 in the developing mouse forebrain (Lagutin et al, 2003), and the Wnt/β-catenin signaling pathway mediates mouse lens development (Smith et al, 2005). To determine whether alterations in the Wnt/β-catenin pathway influenced lens formation in Six3-mutant embryos, we used the available Bat-Gal reporter mouse that expresses lacZ in response to activation of the canonical Wnt pathway (Maretto et al, 2003). Control and Six3-mutant E9.5 and E10.5 PLE isolated from crosses of Bat-Gal and Six3f/Δ;Le-Cre mutant mice were both negative for X-gal staining (data not shown). In addition, no nuclear localization of β-catenin was observed in the SE of mutant embryos (data not shown). These results indicate that the lens phenotypes resulting from the specific removal of Six3 are probably not related to defects in the Wnt/β-catenin pathway. However, the drastically reduced expression of Pax6 and the absence of Sox2 in the type III Six3-mutant PLE indicate that lens induction/specification are defective in these severely affected embryos. Thus, Six3 activity in the PLE is essential during lens induction. The time of Six3 removal determines the severity of the lens phenotype To remove Six3 activity at different developmental stages as efficiently as possible but without compromising its role in forebrain formation (Lagutin et al, 2003), we used the CAGG-CRE-ER mouse strain in which ubiquitous Cre activity is induced by tamoxifen (Hayashi and McMahon, 2002). Floxed Six3 (Six3f/f) females were bred with Six3Δ/w;CAGG-CRE-ER males, and two injections of tamoxifen were given to the pregnant dams at approximately E7.8 and early E8.5 for early Six3 deletion, and at E9.5 and E10.0 for late deletion; embryos were then isolated at E10.5. Both protocols deleted Six3 activity efficiently (Figure 4A′ and G′), although defective forebrain patterning was observed in some of the early-injected embryos, which were then no longer considered for the analysis of the lens. Figure 4.The timing of Six3 deletion is crucial for the lens phenotype. Ubiquitous, early deletion of Six3 via tamoxifen injection of pregnant Six3f/Δ;CAGG-Cre-ER dams at E7.8 and E8.5 (A′–F′) and late deletion via injection at E9.5 and E10.0 (G′–I′) were performed. Mutant embryos were analyzed at E10.5. Only early deletion of Six3 caused a failure in lens specification. (A′) Six3 expression was not detected in the PLE (arrow) or optic vesicle (arrowhead). (A′, B′) The PLE did not thicken and the lens placode did not form. (C′, D′) Defective lens induction and specification was confirmed by the absence of Pax6 and Sox2 from the PLE of the mutant embryos (arrows). (E′) Expression of Meis1 remained in the SE. Although Six3 was deleted ubiquitously and the shape of the optic vesicle was abnormal, neural retina expression of Pax6 (arrowhead in C′), and Chx10 (arrow in F′) remained. (G′–I′) Embryos subjected to late Six3 deletion exhibited normal or slightly small lens pits. Pax6 expression was not affected (arrow in H′), and Sox2 was either not affected or was slightly reduced (I′). (J) Graphic representation of the number of analyzed mutant embryos with early or late Six3 deletion per the observed lens phenotype (normal, small, or no lens pit (LP)). Download figure Download PowerPoint No thickening or invagination of the PLE was seen at least in one side of six of the seven E10.5 mutant embryos in which tamoxifen was administered early (E7.8 and E8.5; Figure 4A′ and B′). Pax6 and Sox2 expression were not detected in the PLE but were observed at lower levels in the optic cup (Figure 4C′ and D′). The expression of FoxE3, sFrp2, and Prox1 was not detected in the mutant PLE (data not shown), but that of Meis1 (Figure 4E′) and Meis2 (data not shown) was unaffected. Thus, lens specification was defective, a phenotype similar to that of the severely affected type III embryos described above when using Le-Cre. Interestingly, although the shape of the developing optic cup was abnormal, typical retinal progenitor markers (Pax6, Sox2, Chx10) were still detected (Figure 4C′, D′ and F′). Lens specification was not obviously affected (i.e., the lens pit was normal or slightly smaller) in E10.5 mutant embryos (n=7/7) in which tamoxifen was administered later (E9.5 and E10.0; Figure 4G′). Pax6 expression appeared normal in the mutant lens pit (Figure 4H′), and that of Sox2 was normal or slightly reduced (Figure 4I′), characteristics reminiscent of those described for the types I and II Six3-mutant lenses when using Le-Cre. A graphic representation of these phenotypes is presented in Figure 4J. To complement these studies, we also examined early deleted (tamoxifen injection at approximately E7.8 and early E8.5) Six3-mutant embryos at around E9.0 (18-somite stage, prior to the upregulation of Sox2 in the PLE). In two independent Six3-mutant embryos (Figure 5A′ and D′), only a few Six3-expressing cells remained in the PLE, and Six3 expression was efficiently deleted in the evaginating optic cup (bilateral differences in the rate of Six3 deletion and severity of the lens phenotype were occasionally observed). In adjacent sections, Pax6 expression was consistently reduced in the mutant PLE (Figure 5B′ and E′), although at different levels; it was almost undetectable in some embryos (Figure 5E′) and reduced in others (Figure 5B′). Normally, low levels of Sox2 expression are detected in the PLE at this early stage (Figure 5C and F); however, Sox2 expression was further reduced in the PLE of one of the analyzed mutant littermates (Figure 5C′) and not detected in the PLE of the second one (Figure 5F′). Our results suggest that the downregulation of Pax6 and consequent lack of upregulation of Sox2 in the PLE results from the removal of Six3 at early stages. Figure 5.Early deletion of Six3 downregulates Pax6 preplacodal expression. Ubiquitous, early deletion of Six3 by injection of tamoxifen at E7.8 and E8.5 in Six3f/Δ;CAGG-Cre-ER mutant embryos analyzed at E9.0 (18-somite stage). (A′, D′) Six3 activity was almost completely removed from the PLE (arrowheads) and optic vesicle. Pax6 (B′, E′) and Sox2 (C′, F′) expression was reduced in the PLE, although with varying efficiency. Download figure Download PowerPoint Together, these results argue that the time of Six3 deletion is critical for the lens phenotype, and early Six3 deficiency is responsible for the failure in lens specification. Early Six3 deletion drastically downregulates Pax6 and Sox2 expression in the PLE, and late Six3 deletion minimally affects their expression. Six3 expression in the PLE precedes that of Pax6 We have now demonstrated that early conditional removal of Six3 from the PLE downregulates Pax6 preplacodal expression and arrests lens formation; the resulting lens phenotype is similar to that of Pax6−/− embryos (Grindley et al, 1995). Therefore, the Six3-promoted lens phenotype may be caused by Six3's direct regulation of preplacodal Pax6 in the head SE. To support this proposal, Six3 expression in the presumptive lens SE must start as early as (if not before) that of Pax6. To test this, we characterized the expression profiles of the two proteins starting at around the 5-somite stage (E8.0). Besides strong expression in the anterior neuroectoderm, Six3 was also detected in the head SE that will become lens ectoderm (Figure 6A); barely detectable levels of Pax6 were seen in the adjacent sections (Figure 6A′). Around the 7- to 8-somite stage, Pax6 become visible in the PLE (Figure 6B′); at around E8.5–9.5, Six3 and Pax6 were both localized in the PLE (Figure 6C and C′), lens placode, and optic vesicle (Figure 6D and D′). Figure 6.Six3 expression in the PLE precedes that of Pax6 and is initially unaffected in small eye-mutant embryos. (A) Six3 is expressed in the PLE (arrowhead) of a wild-type 5-somite-stage embryo. (A′) Pax6 expression is not yet detected in that region. At later stages, Six3 (B–D) and Pax6 (B′–D′) are detected in the PLE (arrowheads) and optic vesicles. Six3 expression in the PLE was examined in small eye (Pax6Sey-1Neu/Sey-1Neu)-mutant embryos. Pax6 was not required for the initial expression of Six3 in the PLE (arrowheads in E′, F′) but was required for its maintenance in the PLE at later stages (arrowheads in G′, H′). Download figure Download PowerPoint Six3 PLE expression at around early E9.5 is initially independent of Pax6; later, it becomes Pax6-dependent (Purcell et al, 2005). The finding that Six3 expression in the head SE precedes that of Pax6 suggests that Six3-mediated upregulation of Pax6 is a necessary early step in the commitment toward a lens fate. Supporting this argument, Six3 expression in the PLE was not overtly affected in 6- or 8-somite-stage Pax6Sey-1Neu/Sey-1Neu-mutant embryos (Figure 6E′ and F′), but was downregulated at later stages (Figure 6G′) and was absent at around E9.5–E10.5 (Figure 6H′). In contrast, Six3 expression in the neural retina was unaffected. We conclude that Six3 activity in the SE precedes that of Pax6, and that Pax6 activity, although not necessary to induce Six3 expression, is later required for Six3 maintenance and upregulation in the PLE. Six3 binds to Pax6 and Sox2 lens enhancers Pax6 deficiency causes a lens phenotype (Grindley et al, 1995) similar to that of the severe type III Six3 mutants. This similarity led us to hypothesize that Six3 directly activates Pax6 expression during early lens morphogenesis (i.e., during lens induction). In addition, it has been suggested that Sox2 is downstream of the Pax6 preplacodal phase (Furuta and Hogan, 1998). Sox2 expression was absent from the PLE of type III Six3-mutant lenses. In this context, Six3 could also directly or indirectly activate Sox2 expression in the PLE. During lens formation, Pax6 is regulated by the EE (Williams et al, 1998; Kammandel et al, 1999; Xu et al, 1999) and SIMO enhancers (Kleinjan et al, 2001). In the case of Sox2, three lens enhancers (N3, N4, L) have been identified in the chick (Uchikawa et al, 2003); however, only N3 and N4 function during lens specification. We have identified a consensus core ATTA motif as the typical Six3 DNA-bi