Max-Planck-Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, D-37077 Göttingen, Germany
* Present address: The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Present address: Sackler Faculty of Medicine, Department of Human Genetics and Molecular Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel
These authors contributed equally to this work
Author for correspondence (e-mail: pgruss{at}gwdg.de)
Accepted 26 June 2002
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SUMMARY |
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Key words: Pax6, Enhancer, Retina, Autoregulation, Axon guidance, Superior colliculus, Axis formation, BF-1, BF-2, Mouse
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INTRODUCTION |
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The winged-helix transcription factors BF-1 (Foxg1 Mouse Genome Informatics) and BF-2 (Foxd2 Mouse Genome Informatics) are among the first factors that mark the distinction between the presumptive nasal and temporal NR as they are already expressed before the onset of neuronal differentiation in the anterior and posterior optic vesicle, respectively (Hatini et al., 1994). Overexpression of BF-1 and BF-2 in chick embryos lead to targeting errors of RGC projections to the tectum (Yuasa et al., 1996
). In mice, the absence of BF-1 function severely affects the development of the ventral NR and appears to lead to a mirror-image duplication of the temporal axis (Huh et al., 1999
). Similarly, the T-box and homeodomain transcription factors Tbx5 and Vax2 become expressed in the dorsal and ventral optic cup, respectively, prior to the onset of retinogenesis (Koshiba-Takeuchi et al., 2000
; Barbieri et a., 1999
). Overexpression of Tbx5 results in dorsalization of the NR and in misrouting of ventral axons (Koshiba-Takeuchi et al., 2000
). Conversely, the absence of Vax2 leads to a dorsalization of the NR in the mouse (Mui et al., 2002
; Barbieri et al., 2002), whereas misexpression of the orthologous Vax in chick embryos disturbed dorsal RGC projections (Schulte et al., 1999
). Null mutants of Vax1, a mouse paralog of Vax2, show optic cup coloboma and optic nerve dysplasia with axon misguidance, which is due to its expression in the very ventral NR and in the optic stalk (Hallonet et al., 1998
; Hallonet et al., 1999
; Bertuzzi et al., 1999
). Furthermore, the homeodomain factors SOHo1 and GH6 were implicated in mediating proper retinotectal projections from nasal RGCs in the chick embryo by controlling the expression of EphA3 (Schulte and Cepko, 2000
).
The paired and homeodomain factor Pax6 is a key member of a highly conserved interactive network of transcription factors implicated in the initiation of eye development (Gruss and Walther, 1992; Gehring and Ikeo, 1999
). Forced expression of Pax6 is sufficient to promote the formation of ectopic eye structures in insects and vertebrates (Halder et al., 1995
; Altmann et al., 1997
; Chow et al., 1999
), while Pax6 null mutations invariably lead to the absence of all functional eye structures (Glaser et al., 1990
; Hill et al., 1991
; Quiring et al., 1994
; Grindley et al., 1995
). Recently, we could provide a link between these generic mechanisms governing retinal formation and the cytogenetic potential of retinal progenitor cells (Marquardt et al., 2001
), demonstrating a continued requirement for Pax6 activity in later retinal development. Moreover, the proper development of distal eye structures, iris, cornea and lens epithelium appears to be highly sensitive to variations in the level of Pax6 activity. Heterozygosity for Pax6 mutations in human and mouse lead various degrees of iris hypoplasia and dysplasia, as well as lens and corneal defects (Jordan et al., 1992
; Glaser et al., 1994
; Hanson et al., 1994
; Collinson et al., 2001
), while mice overexpressing Pax6 display microphthalmia associated with retinal, corneal and iris dysplasia (Schedl et al., 1996
). In the newly formed optic cup, Pax6 is soon observed to be expressed in a marked distalhigh-proximallow gradient, suggesting a role in optic cup patterning. The recent identification of a downstream target of Pax6 expressed in the distalmost region of the optic cup (Bernier et al., 2001
) further supports such a role.
In this study, we demonstrate that the distalhigh-proximallow gradient of Pax6 activity in the optic cup is mediated by a highly conserved intronic enhancer () (Kammandel et al., 1999
) of the Pax6 locus. In the mature NR, this enhancer drives expression in a subset of RGCs that stereotypically send their axonal projections to two concise stripes in both the superior colliculus and LGN. Intriguingly, in a targeted mouse line in which the
-enhancer is deleted (St-Onge et al., 1997
), the expression of the remaining regulatory elements of Pax6 can be mapped to RGCs that send their projections to the central region of the superior colliculus, precisely complementing the area targeted by the
-positive RGCs. Therefore, in the mature NR Pax6 expression appears to be regulated in distinct topographic domains that divide superior colliculus and LGN into two concise areas that receive axonal input from (1) the distal nasal and temporal NR, and (2) the proximal NR. In the optic vesicle and later the optic cup, we found that Pax6 activity was required for the establishment, as well as the maintenance, of the dorsal and nasotemporal characteristics, providing a link between generic mechanisms governing retinal development and the patterning of the optic cup, which ultimately underlie the topographic mapping of retinal axons to the brain.
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MATERIALS AND METHODS |
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Immunohistochemistry on cryosections
The embryos were washed in phosphate-buffered saline (PBS), fixed for 30 minutes fresh in cold 4% PFA/PBS (pH 7.8), washed three times 20 minutes each with PBS, incubated in 30% sucrose/PBS for overnight at 4°C and shock frozen in tissue freezing medium (Jung). Sections of 6-10 µm were air-dried for 30 minutes and stored at 20°C or 80°C.
For the antibody staining, the sections were washed three times in PBS (5 minutes each), blocked with 1% BSA (IgG-free, Sigma), 0,05% Tween-20 in PBS 30 minutes at room temperature. The primary antibody was diluted in blocking solution and incubated at 4°C overnight (rabbit anti-ß-gal, Cappel, 1:300; monoclonal mouse anti-Pax6, DSHB, 1:20). After three washes in PBS, 5-10 minutes each, the secondary antibody, diluted in blocking solution, was applied for 1 hour (Alexa 568 goat anti rabbit, Molecular Probes, 1:500; FITC goat anti mouse, Southern Biotechnology, 1:60). After again three washes with PBS, counterstaining was performed with Dapi and the sections were embedded with Mowiol.
Histochemical detection of ß-galactosidase activity
For X-gal staining, embryos or tissues were rinsed in PBS, fixed for 0.5 to 1.5 hours in cold 1% formaldehyde, 0.2% glutaraldehyde in wash buffer (0.2% NP-40, 0.1% Na-desoxycholate in PBS), then washed three time for 20 minutes at room temperature in wash buffer and stained up to 3 days at 30°C in 1 mg/ml X-gal/5 mM K3Fe(CN)6/5 mM K4Fe(CN)6/2 mM MgCl2 in wash buffer. The tissues then underwent the standard procedure for paraffin embedding, were sectioned at 7-10 µm and counterstained with Nuclear Fast Red.
For the staining of cryosectioned tissues, the 10 µm sections were washed twice in PBS, postfixed in cold 0.2% glutaraldehyde for 5 min and washed again in PBS. The X-gal staining was then performed at 37°C for 4 hours to overnight in 1 mg/ml X-gal/5 mM K3Fe(CN)6/5 mM K4Fe(CN)6/2 mM MgCl2 in PBS.
In situ hybridization
In situ hybridization on paraffin wax-embedded sections using 35S-labeled antisense RNA probes to detect the transcripts of BF-1 and BF-2 (Hatini et al., 1994) were carried out as previously described (Kessel and Gruss, 1991
). The in situ hybridization of Tbx5 (probe a gift of J. Johnson), Vax1 (Hallonet et al., 1999
) and Vax2 (Barbieri et al., 1999
) were performed as described previously (Marquardt et al., 2001
).
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RESULTS |
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Pax6 activity is required for initiating and upregulating, but not for maintaining retinal expression via the -enhancer
Pax6 has previously been suggested to regulate its own expression. First, Pax6 transcripts were shown to be absent from the head surface ectoderm of Pax6-null mutant embryos (Grindley et al., 1995). Second, gfp reporter gene expression driven by a lens-specific regulatory element of Pax6 was observed to be lost after the Cre-mediated inactivation of Pax6 (Ashery-Padan et al., 2000
). Results from in vitro experiments indicated that Pax6 directly binds and activates the
-enhancer (Schwarz et al., 2000
). However, the relevance of Pax6 activity for the
-enhancer-mediated expression in vivo remained unaddressed. To elucidate whether the
-enhancer becomes activated in the absence of Pax6 activity we crossed a transgene,
-Cre-gfp, expressing gfp and Cre under the control of the
-enhancer (Fig. 1B) (Marquardt et al., 2001
), to mice carrying a targeted Pax6-null mutation (Fig. 1A) (St-Onge et al., 1997
). We failed to detect gfp expression in
-gfp; Pax6/ embryos (Fig. 2C), indicating that the initiation of
-enhancer mediated expression depends on the presence of Pax6 activity.
We next analyzed the impact of Cre-mediated inactivation of Pax6 in the optic cup stage retina on -enhancer mediated activity in
-Cre-gfp; Pax6flox/flox embryos (Marquardt et al., 2001
; Ashery-Padan et al., 2000
). In
-Cre-gfp; Pax6flox/flox embryos Pax6 is eliminated from virtually all cells of the distal nasal and temporal NR at around E10.5 (Marquardt et al., 2001
). In
-Cre-gfp; Pax6flox/flox embryos, gfp expression was detectable in the distal NR up to the day of birth (P0), that is for at least 8 days after loss of Pax6 activity (Fig. 2B) (Marquardt et al., 2001
) (data not shown). However, the gfp expression is significantly reduced in the
-Cre-gfp; Pax6flox/flox NR (Fig. 2B). Interestingly, gfp expression ultimately becomes extinguished in most of the distal NR during the first postnatal days (data not shown). Together, these results indicate that an early phase of Pax6 activity appears to be essential for the initiation of
-enhancer-mediated expression, while Pax6 activity appears not to be strictly required for maintaining
-enhancer activity. However, the significantly reduced gfp expression levels in the
-Cre-gfp; Pax6flox/flox (Fig. 2B) and the
-Cre-gfp; Pax6+/(data not shown) NR indicate that positive Pax6 autoregulation via direct interaction with the
-enhancer might contribute to the steep distalhigh-proximallow gradient of Pax6 activity in the optic cup.
-enhancer directed expression defines a subset of Pax6 positive retinal ganglion cells projecting to two concise areas in the primary visual relay centers
The Pax6 -enhancer continues to direct expression in the postnatal and adult eye, most prominently in the retinal ganglion cells (RGCs) of the distal nasal and temporal NR (Fig. 3C-F). This observation prompted us to map the projections of this subset of Pax6+ RGCs. To this end we created a transgenic mouse line in which the axonal marker gene tau-lacZ (Callahan and Thomas, 1994
; Mombaerts et al., 1996
) was placed under the control of the
-enhancer (Fig. 3I, referred to as
-tau-lacZ). Tau-ß-galactosidase (tau-ß-gal) expression directed by the
-tau-lacZ transgene displays a gradient in the distal NR (Fig. 3B,C) corresponding to the Cre/gfp activity directed by
-Cre-IRES-gfp (see Fig. 1E, Fig. 2A) (Marquardt et al., 2001
).
In newborn (P0) -tau-lacZ mice, the histochemical detection of tau-ß-gal allowed the visualization of axons projecting from distal nasal and temporal NR through the optic chiasm up to the lateral geniculate nucleus (LGN) (Fig. 4A). Around postnatal day 15 (P15), tau-ß-gal was detectable in RGC axons growing into the superior colliculus (SC, Fig. 4B,E,G). In the superior colliculus of
-tau-lacZ mice, the tau-ß-gal+ axons terminated in a highly stereotypic fashion within two concise areas, forming two sickle-shaped domains in the rostral and caudal superior colliculus, respectively (Fig. 4E,G). Virtually no tau-ß-gal+ RGC axons could be observed to terminate within the central region of the superior colliculus in all
-tau-lacZ analyzed individuals (Fig. 4E,G) and the labeled axons invariably followed the projection pattern outlined above. In the LGN, tau-ß-gal+ RGC projections were detected to terminate in a similar fashion, marking two stripes in the rostroventral and caudodorsal LGN, respectively (Fig. 4E,F,H). In coronal sections through the LGN, an additional condensed area with terminating projections were detected in the central LGN (Fig. 4H: bs), possibly corresponding to the binocular segment which receives projections from ipsilateral ventrotemporal RGCs (Dräger and Olsen, 1980
; Godement et al., 1984
). Thus, the
-tau-lacZ transgene line provides a valuable tool to study retinal projection defects in mutant models.
Additionally, the highly ordered pattern of projections of distal Pax6+ RGCs marked by the -tau-lacZ transgene revealed an intriguing complexity in the regulation of retinal Pax6 expression, which was further explored as described below. The
-tau-lacZ transgene furthermore provides a valuable transgenic tool to study projections in mutant models for axonogenesis in the optic tract, potentially complementing limitations inherent to conventional antero- and retrograde dye tracings.
Pax6 expression is regulated in two complementary topographic domains in the mature retina
The sustained expression of tau-ß-gal in the postnatal and adult retina of -tau-lacZ animals suggested a role of the
-enhancer in regulating Pax6 expression in mature RGCs of the distal NR. To examine the late function of this regulatory element, we analyzed the expression of ß-gal in the postnatal and adult Pax6lacZ/+ retina, in which the
-enhancer is deleted by the insertion of lacZ as mentioned above (see Fig. 1A). Like the normal distribution of endogenous Pax6 expression in the adult retina, ß-gal expression in the P20 Pax6lacZ/+ retina was mainly confined to the ganglion cell layer (Fig. 5B,F). However, unlike endogenous Pax6, ß-gal expression in the P20 Pax6lacZ/+ retina was virtually absent in distal RGCs (Fig. 5B,E) compared with the proximal NR (Fig. 5B,F). This expression pattern was precisely complemented in the retina of
-tau-lacZ animals (Fig. 5A,B, inset), where high levels of tau-ß-gal expression were present in the distal retina (Fig. 5C), while the expression sharply decreased towards the optic nerve head (Fig. 5D).
The high levels of ß-gal activity in the retina of Pax6lacZ/+ animals allowed us to follow axonal projections from RGCs up to the superior colliculus (Fig. 5H,J). Interestingly, the ß-gal+ axons in Pax6lacZ/+ animals terminated into the central region of the superior colliculus, leaving out the most anterior and posterior regions (Fig. 5H,J). The comparison of the labeled optic tract projections in P20 Pax6lacZ/+ and -tau-lacZ transgenic animals revealed that the ß-gal+ axons in Pax6lacZ/+ terminate exactly within the gap left by the sickle-shaped tau-ß-gal+ stripes observed in the anterior and posterior superior colliculus of
-tau-lacZ animals (compare Fig. 5G,H with I,J).
To examine whether the absence of ß-gal+ projections in the anterior and posterior superior colliculus in the Pax6lacZ/+ mice is not a consequence of possible misrouting of the distal RGC axons, we studied the ß-gal staining pattern of -tau-lacZ in the background of Pax6 heterozygous small eye mutant mice (
-tau-lacZ; Pax6Sey/+). As illustrated in the insets of Fig. 5B,H,J, the tau-ß-gal+ axons in
-tau-lacZ; Pax6Sey/+ embryos essentially displayed the same innervation pattern as in
-tau-lacZ; Pax6+/+ mice. However, a significant reduction in the number, but not in the intensity of staining of the tau-ß-gal+ axons could be observed (Fig. 5H, inset), which might be attributable to the hypocellular appearance of the distal Pax6Sey/+ retina (data not shown). We conclude therefore that Pax6 activity in the retina is specifically regulated in distinct non-overlapping RGC subpopulations that parcellate the primary visual centers into two complementary topographic domains.
Establishment and maintenance of nasotemporal and dorsal patterning of the retina requires Pax6
The presence of a regulatory element promoting high levels of Pax6 activity in the distal nasal and temporal NR prompted us to address whether Pax6 is required for patterning of the nasotemporal axis in the retina. BF-1 and BF-2 are among the first factors to mark the distinction between the future nasal and temporal retina and become expressed in the nasal and temporal optic vesicle at around E9, respectively (Hatini et al., 1994). We first examined the expression of BF-1 and BF-2 in Pax6-null mutant (Pax6/) embryos by in situ hybridization. Both BF-1 and BF-2 fail to be expressed in the optic vesicles of Pax6/ embryos (Fig. 6C,D), while high levels could still be observed in tissues outside the eye, such as telencephalon and head mesenchyme (Fig. 6C,D). By contrast, in E12.5 wild-type embryos BF-1 and BF-2 expression can be detected in the nasal and temporal NR, respectively (Fig. 6A,B). This observation therefore suggests that Pax6 activity in the optic vesicle is required for initiating the expression of determinants of the nasotemporal axes of the retina.
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These results indicate that Pax6 is required, first, for initiating the expression of factors instrumental in establishing the nasotemporal axis of the retina and, second, for maintaining the expression of nasal and temporal determinants in the NR.
In order to determine the specificity of the failure in establishment of nasotemporal characteristics that is due to the loss of Pax6 activity, we then also examined the expression of dorsoventral markers in the NR. Interestingly, Tbx5, which is normally at E12.5 only expressed in the dorsal NR (Fig. 6G) (Koshiba-Takeuchi et al., 2000), was not detectable in the Pax6 mutant optic vesicle (Fig. 6I). Furthermore, the ventral NR marker Vax1 (Fig. 6H) (Hallonet et al., 1998
) displayed strong expression throughout the dorsoventral extent of the Pax6/ optic vesicle. Additionally, another ventral NR determinant, Vax2, was weakly expressed in the entire optic vesicle (Barbieri et al., 1999
) (data not shown). Together, these data suggest that in the optic vesicle of Pax6-null mutants, ventral retinal characteristics expand at the expense of dorsal and nasotemporal characteristics, consequently leading to a failure in the specification of the nasotemporal and dorsoventral retinal axes.
The Pax6 null mutant optic vesicle expresses markers, such as Chx10, Mitf and Trp2 in regionally defined areas that indicate the appropriate specification of NR and pigmented epithelium progenitor domains (N. B., T. M. and P. G., unpublished) (Grindley et al., 1995). Furthermore, in the
-Cre; Pax6flox/flox retina, retinal progenitor cell characteristics are maintained and neuronal differentiation occurs (Marquardt et al., 2001
). Therefore, in both cases the loss of nasal, temporal (as well as dorsal) retinal determinants is apparently not due to a general failure in retinal development, but rather reflects a direct requirement for Pax6 activity.
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DISCUSSION |
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The dynamic expression of Pax6 during embryogenesis necessarily requires complex regulatory mechanisms. Recently, several distinct regulatory elements have been identified that are able to recapitule Pax6 expression in ectodermally derived eye structures, in the distal NR (Kammandel et al., 1999; Xu et al., 1999
), as well as in the telencephalon and endocrine pancreas (Kammandel et al., 1999
); together revealing an intriguing modular organization of the Pax6 locus. The function of the evolutionary highly conserved retinal
-enhancer (Kammandel et al., 1999
; Xu et al., 1999
) provided a puzzle, as its activity starting E10.5 is initiated well after the onset of endogenous Pax6 and only in a subdomain of retinal Pax6 expression.
In this study, we provide evidence that Pax6 activity controlled by the -enhancer contributes to the establishment of the distalhigh-proximallow gradient of Pax6 expression in the NR and for maintaining Pax6 expression in the distal NR (Fig. 1C-E, Fig. 2), while the remaining regulatory element/s of Pax6 continue to drive expression in the proximal region of the optic cup (see Fig. 5). Recently, a regulatory element residing downstream of the Pax6 gene has been isolated, apparently driving the early aspects of Pax6 expression from E8.5 in the optic vesicle and later the neural and pigmented retina (Lauderdale et al., 2000
; Kleinjan et al., 2001
). Further small regulatory sequences that drive specific aspects of retinal Pax6 expression have been identified, one of them driving expression in the developing photoreceptor cells (Xu et al., 1999
). Therefore, a complex interplay of distinct modularly organized regulatory elements scattered across the entire Pax6 locus appears to underlie the highly dynamic pattern of Pax6 activity during optic vesicle and retinal development.
Assuring sufficient Pax6 levels in the distal retina
The presented results strongly suggest that a main function of the -enhancer consists in mediating high levels of Pax6 activity in the distal NR (Fig. 7A). The highest level of the Pax6 gradient in the NR marks the future iris epithelium (see Fig. 1). The iris appears to be especially sensitive to variations in Pax6 levels, as both reduction (Jordan et al., 1992
; Glaser et al., 1994
) or increase (Schedl et al., 1996
) in Pax6 dose can result in severe iris malformations or absence of iris structures. These observations appear to reflect a cell-autonomous requirement for Pax6 function.
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A link between retinal determination and the establishment of retinal axes
The jigsaw puzzle like organization of Pax6 expression in distal and proximal territories of the postnatal NR (Fig. 5A-F) suggested a more specific role of Pax6 in retinal ganglion cell (RGC) axonogenesis. However, the experimental shallowing of the distalhigh-proximallow Pax6 gradient, by conditional ablation of one Pax6 allele in the distal NR, did not appear to result in gross defects in the targeting of distal RGC axons to the superior colliculus and lateral geniculate nucleus (data not shown). Similarly, the general ability of distal RGC axons to project to their correct target positions is not impaired in Pax6 heterozygotes (Fig. 5B,H,J, insets). Therefore, in contrast to the development of the iris, neither absolute nor relative high Pax6 levels in the distal NR appear to be required for the proper targeting of distal RGC axons. Nevertheless, a hypocellular appearance of the distal NR could be observed in Pax6 heterozygotes. This observation, however, might reflect the improper specification of distalmost NR into inner iris epithelium, resulting in an incomplete separation of both tissues. A significant change in the cellular composition of the distal NR could not be observed in adult Pax6lacZ/+ eyes (data not shown). Further reductions in Pax6 levels are unlikely to reveal a specific impact on RGC axonogenesis, owing to the central function of Pax6 in retinal progenitor cells (Marquardt et al., 2001). The precise relevance of the division of the postnatal optic cup in two complementary Pax6 expression domains with distinct targeting fields in the primary visual center requires further investigation. Nevertheless, these observations underline the complex interconnected nature of the regulatory mechanisms governing retinal development, apparently coordinating diverse processes, such as retinogenesis and axon guidance.
However, it remained unclear how the action of such factors is coupled to the generic mechanisms of retinal determination. We provide evidence that one of the central early retinal determinants, Pax6, mediates the regulation of factors that display restricted localization across the retinal axes. We observed that the expression of BF-1 and BF-2 depends on Pax6 activity (see Fig. 6), implicating a direct role of Pax6 in establishment of the naso-temporal axes (Fig. 7B). The concomitant failure to initiate Tbx5 expression in the absence of Pax6 might be indirectly mediated by the dorsal extension of Vax2, which has recently been shown to be a potent repressor of Tbx5 in the chick retina (Koshiba-Takeuchi et al., 2000). Tbx5 overexpression appears to specifically affect dorsoventral, but not naso-temporal retinal axon pathfinding (Koshiba-Takeuchi et al., 2000
), while BF-1 expression seems to be unaffected in Tbx5-null mutants (Mui et al., 2002
; Barbieri et al., 2002). Therefore, the absence of BF-1 and BF-2 expression in the Pax6 null mutant optic vesicle seems to be directly due to the lack of Pax6 rather than indirectly via repression by the expanded domain of Vax2 activity. These results are summarized schematically in Fig. 7B. The relevance of Pax6 for the maintenance of dorsal and/or ventral retinal characteristics, i.e. Tbx5 or Vax2 expression, remains to be elucidated, as in our
-Cre;Pax6flox/flox model the ventral and dorsalmost retinal domains are omitted by Cre activity (Marquardt et al., 2001
). Remarkably, a similar ventralization in the absence of Pax6 activity was already observed in several neural tissues, including the telencephalon (Stoykova et al., 2000
), diencephalon (Grindley et al., 1997
; Warren and Price, 1997
), spinal cord (Ericson et al., 1997
) and pituitary gland (Kioussi et al., 1999
), indicating a general role of Pax6 in dorsoventral patterning of the neural tube.
These observations moreover provide an intriguing link between the generic mechanisms of retinal determination, as well as retinogenesis and the establishment of topographic organization in the visual system. Such a link, however, raises the issue of how a transcriptional activator expressed throughout the entire optic vesicle and later the NR might control the expression of factors that have to be confined to only one particular axis. The inactivation of BF-1 has been reported to result in ectopic expression of the temporal marker BF-2 in the nasal NR (Huh et al., 1999). This observation suggests that BF-1, presumably via its function as a transcriptional repressor (Yao et al., 2001
), mediates nasal NR characteristics by actively suppressing temporal NR determinants that otherwise would become activated throughout the optic cup. The mechanisms leading to the initial restriction of these factors, however, remain to be elucidated, but presumably involves signaling originating from periocular tissue.
These findings furthermore highlight the fact that during embryonic development, the same transcription factor is commonly used during different sequential stages, often contributing to vastly different developmental outcomes. Although initial steps have recently been accomplished in elucidating how a single factor can sequentially govern the specification of two different cell fates (reviewed by Marquardt and Pfaff, 2001), the deciphering of the underlying transcriptional mechanisms remains a major problem in developmental biology.
Note added in proof
Note the name change of Nicole Andrejewski to Nicole Bäume.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Altmann, C. R., Chow, R. L., Lang, R. A. and Hemmati-Brivanlou, A. (1997). Lens induction by Pax-6 in Xenopus laevis. Dev. Biol. 185, 119-123.[Medline]
Ashery-Padan, R., Marquardt, T., Zhou, X. and Gruss, P. (2000). Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev. 14, 2701-2711.
Barbieri, A. M., Lupo, G., Bulfone, A., Andreazzoli, M., Mariani, M., Fougerousse, F., Consalez, G. G., Borsani, G., Beckmann, J. S., Barsacchi, G. et al. (1999). A homeobox gene, Vax2, controls the patterning of the eye dorsoventral axis. Proc. Nat. Acad. Sci. USA 96, 10729-10734.
Bernier, G., Panitz, F., Zhou, X., Hollemann, T., Gruss, P. and Pieler, T. (2000). Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos. Mech. Dev. 93, 59-69.[Medline]
Bernier, G., Vukovich, W., Neidhardt, L., Herrmann, B. G. and Gruss, P. (2001). Isolation and characterization of a downstream target of Pax6 in the mammalian retinal primordium. Development 128, 3987-3994.
Bertuzzi, S., Hindges, R., Mui, S. H., OLeary, D. D. and Lemke, G. (1999). The homeodomain protein vax1 is required for axon guidance and major tract formation in the developing forebrain. Genes Dev. 13, 3092-3105.
Callahan, C. A. and Thomas, J. B. (1994). Tau-beta-galactosidase, an axon-targeted fusion protein. Proc. Natl. Acad. Sci. USA 91, 5972-5976.[Abstract]
Cheng, H. J., Nakamoto, M., Bergemann, A. D. and Flanagan, J. G. (1995). Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82, 371-381.[Medline]
Chow, R. L., Altmann, C. R., Lang, R. A. and Hemmati-Brivanlou, A. (1999). Pax6 induces ectopic eyes in a vertebrate. Development 126, 4213-4222.
Collinson, J. M., Quinn, J. C., Buchanan, M. A., Kaufman, M. H., Wedden, S. E., West, J. D. and Hill, R. E. (2001). Primary defects in the lens underlie complex anterior segment abnormalities of the Pax6 heterozygous eye. Proc. Natl. Acad. Sci. USA 98, 9688-9693.
Dräger, U. C. and Olsen, J. F. (1980). Origins of crossed and uncrossed retinal projections in pigmented and albino mice. J. Comp. Neurol. 191, 383-412.[Medline]
Drescher, U., Kremoser, C., Handwerker, C., Loschiner, J., Noda, M. and Bonhoeffer, F. (1995). In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands of Eph receptor trypsine kinases. Cell 82, 359-370.[Medline]
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169-180.[Medline]
Feldheim, D. A., Kim, Y. I., Bergmann, A. D., Frisen, J., Barbacid, M. and Flanagan, J. G. (2000). Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron 25, 563-574.[Medline]
Gehring, W. J. and Ikeo, K. (1999). Pax 6: mastering eye morphogenesis and eye evolution. Trends Genet. 15, 371-377.[Medline]
Glaser, T., Lane, J. and Housman, D. (1990). A mouse model of the aniridia-Wilms tumor deletion syndrome. Science 250, 823-827.[Medline]
Glaser, T., Jepeal, L., Edwards, J. G., Young, S. R., Favor, J. and Maas, R. L. (1994). PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat. Genet. 7, 463-471.[Medline]
Godement, P., Salaun, J. and Imbert, M. (1984). Prenatal and postnatal development of retinogeniculate and retinocillicular projections in the mouse. J. Comp. Neurol. 230, 552-575.[Medline]
Goodhill, G. J. and Richards, L. J. (1999). Retinotectal maps: molecules, models and misplaced data. Trends Neurosci. 22, 529-534.[Medline]
Grindley, J. C., Davidson, D. R. and Hill, R. E. (1995). The role of Pax-6 in eye and nasal development. Development 121, 1433-1442.
Grindley, J. C., Hargett, L. K., Hill, R. E., Ross, A. and Hogan, B. L. (1997). Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon. Mech. Dev. 64, 111-126.[Medline]
Gruss, P. and Walther, C. (1992). Pax in development. Cell 69, 719-722.[Medline]
Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788-1792.[Medline]
Hallonet, M., Hollemann, T., Wehr, R., Jenkins, N. A., Copeland, N. G., Pieler, T. and Gruss, P. (1998).Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain. Development 125, 2599-2610.
Hallonet, M., Hollemann, T., Pieler, T. and Gruss, P. (1999). Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system. Genes Dev. 13, 3106-3114.
Hanson, I. M., Fletcher, J. M., Jordan, T., Brown, A., Taylor, D., Adams, R. J., Punnett, H. H. and van Heyningen, V. (1994). Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters anomaly. Nat. Genet. 6, 168-173.[Medline]
Hatini, V., Tao, W. and Lai, E. (1994). Expression of winged helix genes, BF-1 and BF-2, define adjacent domains within the developing forebrain and retina. J. Neurobiol. 25, 1293-1309.[Medline]
Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V. (1991). Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature 354, 522-525.[Medline]
Hogan, B., Beddington, R., Constantini, G. and Lacy, E. (1994). Manipulating the Mouse Embryo, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Holder, N. and Klein, R. (1999). Eph receptors and ephrins: effectors of morphogenesis. Development 126, 2033-2044.
Huh, S., Hatini, V., Marcus, R. C., Li, S. C. and Lai, E. (1999). Dorsal-ventral patterning defects in the eye of BF-1-deficient mice associated with a restricted loss of shh expression. Dev. Biol. 211, 53-63.[Medline]
Hyatt, G. A., Schmitt, E. A., Marsh-Armstrong, N., McCaffery, P., Drager, U. C. and Dowling, J. E. (1996). Retinoic acid establishes ventral retinal characteristics. Development 122, 195-204.
Jordan, T., Hanson, I., Zaletayev, D., Hodgson, S., Prosser, J., Seawright, A., Hastie, N. and van Heyningen, V. (1992). The human PAX6 gene is mutated in two patients with aniridia. Nat. Genet. 1, 328-332.[Medline]
Kammandel, B., Chowdhury, K., Stoykova, A., Aparicio, S., Brenner, S. and Gruss, P. (1999). Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. Dev. Biol. 205, 79-97.[Medline]
Kessel, M. and Gruss, P. (1991). Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67, 89-104.[Medline]
Kioussi, C., OConnell, S., St-Onge, L., Treier, M., Gleibermann, A. S., Gruss, P. and Rosenfeld, M. G. (1999). Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development. Proc. Nat. Acad. Sci. USA 96, 14378-14382.
Kleinjan, D. A., Seawright, A., Schedl, A., Quinlan, R. A., Danes, S. and van Heyningen, V. (2001). Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6. Hum. Mol. Genet. 10, 2049-2059.
Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose,T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K. and Ogura, T. (2000). Tbx5 and the retinotectum projection. Science 287, 134-137.
Lauderdale, J. D., Wilensky, J. S., Oliver, E. R., Walton, D. S. and Glaser, T. (2000). 3' deletions cause Aniridia by preventing PAX6 gene expression. Proc. Natl. Acad. Sci. USA 97, 13755-13759.
Lund, R. D., Lund, J. S. and Wise, R. P. (1974). The organization of the retinal projection to the dorsal lateral geniculate nucleus in pigmented and albino rats. J. Comp. Neurol. 158, 383-404.[Medline]
Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F. and Gruss, P. (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105, 43-55.[Medline]
Marquardt, T. and Pfaff, S. L. (2001). Cracking the transcriptional code for cell specification in the neural tube. Cell 106, 651-654.[Medline]
Marquardt, T. and Gruss, P. (2002). Generating neuronal diversity in the retina: one for nearly all. Trends Neurosci. 25, 32-38.[Medline]
Mombaerts, P., Wang, F., Dulac, C., Chao, S. K., Nemes, A., Mendelsohn, M., Edmondson, J. and Axel, R. (1996). Visualizing an olfactory sensory map. Cell 87, 675-686.[Medline]
Mui, S. H., Hindges, R., OLeary, D. D., Lemke, G. and Bertuzzi, S. (2002). The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal axes of the eye. Development 129, 797-804.
OLeary, D. D. and Wilkinson, D. G. (1999). Eph receptors and ephrins in neural development. Curr. Opin. Neurobiol. 9, 65-73.[Medline]
OLeary, D. D. M., Yates, P. A. and McLaughlin, T. (1999). Molecular development of sensory maps: representing sights and smells in the brain. Cell 96, 255-269.[Medline]
Oliver, G., Loosli, F., Koster, R., Wittbrodt, J. and Gruss, P. (1996). Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech. Dev. 60, 233-239.[Medline]
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265, 785-789.[Medline]
Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani, T., Iemura, S., Yamamoto, T. S., Ueno, N. and Noda, M. (2001). Ventroptin: a BMP-4 antagonist expressed in a double-gradient pattern in the retina. Science 293, 111-115.
Schedl, A., Ross, A., Lee, M., Engelkamp, D., Rashbass, P., van Heyningen, V. and Hastie, N. D. (1996). Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities. Cell 86, 71-82.[Medline]
Schulte, D. and Cepko, C. L. (2000). Two homeobox genes define the domain of EphA3 expression in the developing chick retina. Development 127, 5033-5045.
Schulte, D., Furukawa, T., Peters, M. A., Kozak, C. A. and Cepko, C. L. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24, 541-553.[Medline]
Schwarz, M., Cecconi, F., Bernier, G., Andrejewski, N., Kammandel, B., Wagner, M. and Gruss, P. (2000). Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127, 4325-4334.
Simon, D. K. and OLeary, D. D. M. (1992). Development of topographic order in the mammalian retinocollicular system. J. Neurosci. 12, 1212-1232.[Abstract]
St-Onge, L., Sosa-Pineda, B., Chowdhury, K., Mansouri, A. and Gruss, P. (1997). Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. Nature 387, 406-409.[Medline]
Stoykova, A., Treichel, D., Hallonet, M. and Gruss, P. (2000). Pax6 modulates the dorsoventral patterning of the mammalian telencephalon. J. Neurosci. 20, 8042-8050.
Vetter, M. L. and Brown, N. L. (2001). The role of basic helix-loop-helix genes in vertebrate retinogenesis. Semin. Cell. Dev. Biol. 6, 491-498.
Wagner, E., McCaffery, P. and Drager, U. C. (2000). Retinoic acid in the formation of the dorsoventral retina and its central projections. Dev. Biol. 222, 460-470.[Medline]
Walther, C. and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113, 1435-1449.[Abstract]
Warren, N. and Price, D. J. (1997). Roles of Pax-6 in murine diencephalic development. Development 124, 1573-1582.
Xu, P. X., Zhang, X., Heaney, S., Yoon, A., Michelson, A. M. and Maas, R. L. (1999). Regulation of Pax6 expression is conserved between mice and flies. Development 126, 383-395.
Yao, J., Lai, E. and Stifani, S. (2001) The winged-helix protein brain factor 1 interacts with groucho and hes proteins to repress transcription. Mol. Cell. Biol. 6, 1962-1972.
Yuasa, J., Hirano, S., Yamagata, M. and Noda, M. (1996). Visual projection map specified by topographic expression of transcription factors in the retina. Nature 382, 632-635.[Medline]
Zuber, M. E., Perron, M., Philpott, A., Bang, A. and Harris, W. A. (1999). Giant eyes in Xenopus laevis by overexpression of XOptx2. Cell 98, 341-352.[Medline]