1 Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, 37077 Göttingen, Germany
2 Max Planck Institute of Immunology, Stübeweg 51, D-79108, Freiburg, Germany
*Author for correspondence (e-mail: pgruss{at}gwdg.de)
Accepted July 16, 2001
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SUMMARY |
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Key words: Necab, Pax6, Retina, Eye development, Mouse
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INTRODUCTION |
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In rodents, in vitro grafting experiments and conditional mutagenesis showed a cell-autonomous requirement for Pax6 during lens placodes formation (Fujiwara et al., 1994; Ashery-Padan et al., 2000). Chimera aggregation studies also revealed a cell-autonomous function for Pax6 during neural ectoderm development, although Pax6 mutants do form an optic vesicle (Hill et al., 1991; Grindley et al., 1995; Quinn et al., 1996; Collinson et al., 2000). In addition, recent experiments using conditional mutagenesis have revealed that Pax6 is also required for the multipotent state of retinal progenitor cells (Marquardt et al., 2001). Although much is known about the role of Pax6 during eye development in the mouse, few genes have been shown to be dependent on Pax6 activity for their expression in the visual system.
We report on the isolation of a novel mouse gene that is genetically downstream of Pax6 in the early developing retina. This gene has been named Necab (Neuronal Ca2+ binding protein; T. C. Sudhof, personal communication). Necab expression is detected in the neural ectoderm of the optic vesicle and in the pre-tectum, where its expression is abolished in Pax6 mutant embryos. Gain-of-function experiments revealed that Pax6 induces ectopic expression of Necab. Remarkably, we also found that Necab misexpression induces ectopic expression of Chx10. Our results show that Necab is genetically downstream of Pax6 in the mouse retinal and forebrain primordium and suggest that it has an activity in regulating gene expression.
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MATERIALS AND METHODS |
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Antibodies
Three synthetic peptides were generated (Ab1, AHRLLREPPPQGRA (amino acids 13-26); Ab2, KPSHAVNESRYGGPT (amino acids 207-221); Ab3, EGQISRLAELIGR (amino acids 252-269). Coupled peptides were injected into rabbits for immunization (Eurogentech, Belgium). Serum from the final bleeding was purified using affinity resin columns, according to the manufacturer recommendations (Immunotech). Eluates were tested at different concentration on paraffin sections by immunohistochemistry using the ABC amplification kit (Vector). Average working concentration for the primary antibodies was 1/50 (second eluate). For immunofluorescence on sections, 17.5 dpc mouse embryos were immersed in cryomatrix solution (Shandon, Pittsburgh) and snap frozen in liquid nitrogen. Embryos were sectioned at 14 µm with a cryostat (Leica), air dried on slides for 10 minutes, washed twice with phosphate-buffered saline (PBS) and fixed for 10 minutes in 4% paraformaldehyde (PFA)/PBS (pH 7.5). After washes in PBS, sections were pre-incubated in 3% bovine serum albumin/0.1% Tween 20/PBS (1 hour), incubated with the primary antibodies overnight at 4°C in the same solution. After washes with PBS, sections were incubated with a diluted (1/1000) goat anti-rabbit secondary antibody (Mobitec) for 1 hour at room temperature, rinsed with PBS and visualized with a fluorescence microscope (Olympus BX60). For the protein-fusion construct, Necab cDNA fragment (clone 9075, amino acids 1-378) was inserted in the pcDNA3.1/V5-His B vector (Invitrogen) in frame with the C-terminal V5/His epitope. COS cells were transfected (FuGENE, Roche) for 24-48 hours with the Necab-V5/His construct, fixed with 4% PFA/PBS (10 minutes at room temperature), washed, incubated with the anti-V5 primary antibody (1/500), washed and incubated with the goat anti-mouse (Mobitec) secondary antibody (1/1000).
In situ hybridization
Embryos were dissected in PBS, fixed overnight in 4% paraformaldehyde at 4°C and embedded in Paraplast (Monoject Scientific). Sections (10 µm) were cut and dried onto chromalum-gelatin slides. All the steps of high-stringency hybridization and washing were carried out as described previously (Kessel and Gruss, 1991) 35S-labelled RNA probe using SP6, T3 or T7 RNA polymerase were done with Boehringer enzyme according to the directive of the company. Exposure time for the radioactive RNA in situ hybridization was 15 days. For double whole-mount in situ hybridization, preparation were hybridized with digoxigenin-labelled and fluorescein-labelled RNA probes, first visualized with alkaline phosphatase-coupled anti-digoxigenin antibody (1/2000) (Boehringer) and NBT/BCIP substrate (Boehringer) at pH 9.5. To destroy the first antibody, embryos were treated with 0.1M glycine/HCl pH 2.2 for 15 minutes and fixed overnight in 4% PFA/PBS at 4°C. For the second colour reaction, embryos were incubated with an anti-fluorescein-AP antibody (1/2000) (Roche) and treated with the Fast Red TR/NAPHTHOL AS-MX phosphate substrate (Sigma). The DNA fragments used to generate the riboprobe were as follows: 1.4 kb Six3 cDNA (Oliver et al., 1995); 1.9 kb Necab cDNA (clone 9066); and 800 bp Pax6 cDNA (Walther and Gruss, 1991). The 1.2 kb Rx cDNA and 1.4 kb Lhx2 cDNA were amplified by RT-PCR based on the available sequences in the database and cloned into the PCRII vector (Clontech). The 800 bp Chx10 cDNA fragment was a gift from Roderick McInnes (Toronto) and the 900 bp Otx2 cDNA was a gift from Thomas Theil (Düsseldorf). For cryosectioning, embryos were cryoprotected in 30% sucrose/PBS overnight at 4°C, embedded in cryomatrix solution and snap-frozen in liquid nitrogen. Specimens were cut using a cryostat (Leica) at 14 µm, air dried, washed twice in PBS and mounted in 80% glycerol/PBS solution.
Embryos culture and electroporation
The full-length cDNA fragment of mouse Pax6 (Walther and Gruss, 1991), mouse Six3 (Oliver et al., 1995) and mouse Necab (clone 9075) were inserted directly into the eucaryotic expression vector pCS2+. Large DNA preparations were purified using the Maxi-prep filter column system (Qiagen), treated with phenol/chloroform/isoamyl alcohol (pH 8.0), chloroform/isoamyl alcohol, precipitated and washed with 70% ethanol. The DNA was resuspended in PBS at a concentration of 5 µg/µl. Mouse embryos from the NMRI strain were isolated at embryonic stages 8.5, according to the time of the vaginal plug. Dissection was initially performed in PBS to isolate the conceptus. The embryos were transferred in M2 medium (Sigma) supplemented with 10% foetal calf serum (FCS)/10 mM glucose at room temperature. The deciduas and Reicherts membrane were carefully removed, keeping the yolk sac and the ectoplacental cone intact. Embryos were transferred into a microcuvette containing PBS and injected (Eppendorf injector, model 5242) with a capillary needle into the midbrain region. The embryos were electroporated (three pulses at 35 Volts) using a generator power supply (BTX, ECM 8300). Injected embryos were transferred into a 6 wells petri dish for cell culture containing 3 ml/well of 100% FCS/10 mM glucose and cultured for 1 to 6 hours into a 5% CO2 incubator at 37°C. Cultured embryos were transferred into PBS, dissected out of the yolk sac and fixed overnight in 4% PFA/PBS (pH 7.5) at 4°C. Abnormal embryos (abnormal somitogenesis or no beating heart) were discarded. After fixation, embryos were stored at 20°C in 100% methanol. For the culture of the explants, midbrains from 8.5 dpc embryos were isolated in PBS using sharpened tungsten needles, transferred into a microcuvette containing the DNA construct at 1 µg/µl in PBS and pulsed three times (50 mseconds/pulse) at 70 Volts. Explants were mixed with rat collagen (80% rat collagen, 1x modified Eagles medium, 5% FCS, 2% L-glutamine, equilibrated with NaHCO3 at pH 7.5) and placed into a tissue culture net in a 24 wells petri dish. The collagen was allowed to polymerize for 10 minutes at 37°C and the explants were incubated for 24 hours (37°C, 5% CO2) in 1 ml of culture solution (Optimem-Glutamax/F12 (3:1) (Gibco), 10mM glucose, 10% FCS, 1% penicillin/streptavidin). Explants were fixed in 4% PFA/PBS, washed in PBS, dissected out of the collagen polymer and processed for in situ hybridization.
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RESULTS |
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Pax6 can induce ectopic expression of Necab
Previous overexpression experiments in medaka and in Xenopus embryos have revealed that the midbrain region of vertebrates is competent to form retinal tissue when under the influence of Six3, Six6 (Optx2) or Pax6 transcription factors (Loosli et al., 1999; Chow et al., 1999; Bernier et al., 2000). Using DNA electroporation, we tested whether Pax6 could induce ectopic expression of Necab in vivo in the midbrain region of 8.5 dpc mouse embryos. Embryos were electroporated, cultured for 6 hours, and processed for double in situ hybridization (Fig. 5A). Using a DIG-labelled Necab riboprobe (in blue), we detected endogenous expression in the optic vesicles and in the pre-tectum (Fig. 5B). In addition, a few ectopic Necab-expressing cells were observed in the lateral portion of the midbrain. By contrast, the expression domain of the Pax6 transgene (in red) was much broader, but colocalized with Necab-positive cells (Fig. 5C). To see whether this effect was specific to Pax6 activity, embryos were electroporated with a Six3 expression vector, cultured for 6 hours and tested for Necab expression. In all embryos tested (n=12), ectopic expression of Necab was not observed (Fig. 5G-I). To test whether the limited activation of Necab by Pax6 was due to a restriction in cellular competence to express Necab or was simply dependent on time, we electroporated the Pax6 construct in 8.5 dpc mouse midbrain explants and cultivated them for 24 hours. By in situ hybridization, we observed that most of the explants were expressing the transgene (n=22; Fig. 5D). After the second colour reaction, Pax6-positive cells revealed to be also positive for Necab expression (n=22; Fig. 5E). Control explants (n=8), cultured for 24 hours but not electroporated, were negative for both the transgene and for Necab expression (Fig. 5F). We concluded that Pax6 could induce ectopic expression of Necab in vivo but that it requires at least 6 hours to induce this activity.
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DISCUSSION |
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Necab is genetically downstream of Pax6 in the retina
To our knowledge, Necab is the first described gene that coincides with Pax6 expression in the developing optic vesicle and in the pretectum and is Pax6 dependent for its expression. The importance of this discovery is highlighted by the observation that Otx2, Rx, Lhx2 and Six3 expression is not affected by the Pax6 mutation. A previous report has shown that the expression of another homeobox gene expressed in the optic primordium, Six6 (Optx2), is also Pax6 independent (Jean et al., 1999). The hypothesis that Necab is strictly dependent on Pax6 for its expression in the optic vesicle is also strengthened by our result showing that Six3 misexpression could not induce ectopic expression of Necab under the same experimental conditions, showing that Six3 alone is not sufficient for Necab activation. However, Pax6 cannot be the only transcription factor that regulates Necab activity as Necab expression is also detected in body areas outside the Pax6 expression domain, such as the midbrain and the trigeminal and spinal ganglia (not shown).
Necab can induce gene expression
We showed that ectopic expression of Necab is sufficient to induce the expression of Chx10. This result correlates well with the relatively earlier expression of Necab in the developing retina and suggests that Necab might regulate Chx10 expression. However, Necab null mouse mutants are not microphthalmic (G. B. and P. G., unpublished) suggesting either functional redundancy with the other Necab family members or that Necab has a more subtle function during eye development. In addition, care should be taken in deciphering direct genetic interaction between genes while using overexpression experiments, as eye-specific genes form complex regulatory network, as demonstrated in the fly, the fish and the frog. Regardless of this, our results demonstrate that Necab misexpression can induce the transcription of a retina specific gene in vivo. How this is achieved at the molecular level is presently not known. Immunolocalization experiments revealed that Necab is mainly present in the cytoplasm. Interestingly, Eya1, Eya2 and Eya3 genes, the mouse orthologues of eye absent in Drosophila, also encode cytoplasmic proteins (Xu et al., 1997; Ohto et al., 1999). Co-transfection experiments revealed that Eya proteins could be translocated into the nucleus by direct interaction with Six2, Six4 and Six5 proteins (Ohto et al., 1999). Although Eya proteins are mainly cytoplasmic and do not have a DNA-binding domain, overexpression of eya alone in Drosophila has been shown to induce ectopic eye formation (Bonini et al., 1997). It is therefore possible that a similar mechanism applies for Necab, if it could act as a transcriptional co-activator. However, many other options exist that do not require nuclear translocation of Necab in order to induce gene transcription. For example, the cytoplasmic Ca2+-sensitive protein calcineurin can regulate gene transcription by inducing the nuclear translocation of NF-AT transcription complex via its phosphatase activity; see review by Crabtree (Crabtree, 2001). Similarly, RNA injections in Xenopus embryos of the Wnt transmembrane receptor frizzled (Xfz3) resulted in ectopic gene expression and in the formation of ectopic eyes, showing that signal transduction pathways can regulate gene expression during eye development (Rasmussen et al., 2001).
Necab is part of a novel gene family
Necab is part of a novel gene family that is conserved in mammals. The gene family is composed of three members, all containing a putative EF-hand Ca2+-binding domain. This Ca2+-binding domain appears to be functional (T. C. Sudhof, personal communication). Although Necab expression was restricted to the nervous system and some epithelial derivatives (e.g. the olfactory placodes) by in situ hybridization, we also observed strong expression in the dermomyotome with our polyclonal antibodies, suggesting crossreactivity with the other Necab family members. It is thus likely that one of the Necab genes is involved in early myogenesis. Characterization of the expression pattern of the other members should highlight possible functional redundancies of this gene family during development.
In conclusion, we have reported on the isolation of a novel gene expressed in the mouse optic vesicle using a conventional large-scale in situ screen. This gene is a component of the genetic cascade governed by Pax6 during eye formation. In the near future, the combination of whole genome projects and of DNA chip microarray analysis should lead to the identification of the complete set of genes regulated by Pax6 during eye morphogenesis. However, functional characterization of each of these genes will be required in order to decipher the molecular mechanisms of Pax6 activity in the developing eye.
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ACKNOWLEDGMENTS |
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