1 Division of Developmental Biology and Department of Ophthalmology, Childrens Hospital Research Foundation, 3333 Burnet Avenue Cincinnati, OH 45229, USA
2 Division of Human and Molecular Genetics, Childrens Research Institute, 700 Childrens Drive, Columbus, OH 43205, USA
3 The Neurosciences Institute, 10640 John Jay Hopkins Drive, San Diego, CA 92121, USA
*Author for correspondence (e-mail: richard.lang{at}chmcc.org)
Accepted 30 April 2002
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SUMMARY |
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Key words: Bmp, Mouse, Lens, Alk6
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INTRODUCTION |
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One element of lens polarization is the differentiation of the lens fiber cells that express crystallin proteins and impart the features of transparency and refractility (McAvoy, 1978). Evidence that fiber cell differentiation signals have their origin in the retinal cup comes from experiments showing that rotated mouse lenses are only able to repolarize in the presence of retinal tissue (Yamamoto, 1976
). Several signaling pathways have been implicated in the regulation of lens polarity. These include the Fgf signaling pathway, which is important for fiber cell differentiation (Chow et al., 1995
; McAvoy and Chamberlain, 1989
; Robinson et al., 1995a
; Robinson et al., 1995b
; Stolen and Griep, 2000
), and the Igf1 pathway that can regulate fiber cell differentiation in the chick (Beebe et al., 1980
; Beebe et al., 1987
) and epithelial cell proliferation in the mouse (Shirke et al., 2001
). Involvement of TGFß ligands in lens development has recently been uncovered by the attenuated fiber cell differentiation phenotype observed when a dominant-negative TGFß receptor is expressed in the lens (de Iongh et al., 2001
).
Several bone morphogenetic protein (Bmp) family ligand genes are expressed during early development of the eye. These include Bmp4 and Bmp7 (Furuta and Hogan, 1998; Wawersik et al., 1999
), each of which is believed to play an important early role. Bmp7 has a role in lens induction (Wawersik et al., 1999
) where it cooperates with Fgf receptor signaling (Faber et al., 2001
). Bmp4 has also been implicated in lens development and differentiation as it is genetically upstream of Sox2 (Wawersik et al., 1999
), a transcription factor that regulates expression of crystallin genes (Kamachi et al., 2001
). The Bmp4 transcript is present in both the presumptive lens and presumptive retina but is expressed predominantly in the dorsal optic cup as primary fiber cell differentiation begins at E11.5 (Furuta and Hogan, 1998
). Within the lens lineage, Bmp7 is expressed in the presumptive lens ectoderm, lens pit and lens vesicle but is downregulated thereafter (Wawersik et al., 1999
). Based on characterized expression patterns (of Gdf6, for example) (Chang and Hemmati-Brivanlou, 1999
), it might be expected that many other Bmp family ligands will have a role in different aspects of eye development. Bmp signaling occurs by means of ligand binding a primary (type 2) receptor, a complex formation with a transducer, a (type 1) receptor and consequent phosphorylation of Smad proteins (Massagué, 1998
; Piek et al., 1999
). Three known type 2 receptors [Bmpr2, Actr2a (Acvr2 Mouse Genome Informatics) and Actr2b (Acvr2b Mouse Genome Informatics)] and four known type 1 receptors [Actr1 (Acvr1 Mouse Genome Informatics; also known as Alk2), Bmpr1a (also known as Alk3), Bmpr1b (also known as Alk6), Actr1b (Acvr1b Mouse Genome Informatics)] are expressed in the developing eye (Dewulf et al., 1995
; Feijen et al., 1994
; Furuta and Hogan, 1998
; Obata et al., 1999
; Verschueren, 1995
; Yoshikawa et al., 2000
).
In this study, we have investigated the involvement of Bmp signaling in development of the lens. We show that the Bmp binding and inhibition protein noggin can suppress primary fiber cell elongation and lens size in explant culture. We also expressed a dominant negative Bmp type 1 receptor (Bmpr1b; also known as Alk6) in the developing lens in transgenic mice. Both the Pax6 ectoderm enhancer (Kammandel et al., 1999; Williams et al., 1998
; Xu et al., 1999
) and the
A crystallin promoter (Chepelinsky et al., 1985
) were used to drive transgene expression. These mice show defects in the differentiation of primary lens fiber cells, suggesting that Bmp ligands are important for this aspect of lens development. Importantly, we show using anti-Bmpr2 (Gilboa et al., 2000
) and anti-phospho-Smad (Itoh et al., 2001
) antibodies that at embryonic day (E) 12.5, when primary fiber cell differentiation is beginning, equatorial lens cells have Bmp signaling machinery and are responding to Bmp signals. Finally, we show that the primary lens fiber cell differentiation defect is radially asymmetrical, perhaps implying that there are distinct differentiation stimuli active in different quadrants of the eye.
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MATERIALS AND METHODS |
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Histological analysis
Tissues for histological analysis included staged mouse embryos of whole eyes from postnatal animals. Tissue samples were prepared and stained either with Hematoxylin and Eosin (H/E) using conventional methods (Culling et al., 1985). Figures in this paper were prepared digitally using a Sony DKC1000 digital camera and Adobe Photoshop software.
Immunohistochemistry
Staged embryos fixed were fixed in 4% PFA in PBS and immunofluorescently labeled according to conventional methods (Harlow and Lane, 1988). Polyclonal rabbit antisera for MIP26 (Horwitz and Bok, 1987
) was used at a dilution of 1:200, Polyclonal rabbit antisera for
-crystallin (Zigler and Sidbury, 1976
) were both used at a dilution of 1:500. The anti-Bmpr2 (Gilboa et al., 2000
) and anti-phospho-Smad (Itoh et al., 2001
) antibody are affinity-purified rabbit polyclonals from ten Dijke and P. Knaus that were used at a 1:500 and 1:1000, respectively.
3D reconstructions
Reconstructions were created by tracing digital images of 15-20 H/E stained histological sections of wild-type and transgenic lenses in the program Canvas. Canvas tracings were then converted to simple line drawings and imported into the 3-D program FormZ. Individual section tracings were stacked along a z-axis and carefully aligned in both the x and y dimensions. The skin command was then used to mold a skin over the aligned sections. The resulting lenses were then identified as whole objects that could then be rotated in three dimensions so that all sides of the lens could be observed.
Explant culture
Explants of wild-type E10.5 lenses were made following the protocol described elsewhere (Wawersik et al., 1999). Briefly, E10.5 embryos were dissected under a dissecting microscope in PBS at 4°C and the eyes placed in DMEM at 4°C. When eyes from an entire litter had been collected, the presumptive retinal pigmented epithelium was carefully removed with needles. Eyes were then placed in 33% rat-tail collagen (two-thirds DMEM pH 4.0, one third rat-tail collagen, made basic with 0.8M NaCO3 16 µl/200 µl 33% collagen), and left to gel for about 10 minutes. After hardening, the explant collagen gel was immersed in DMEM + 10% FCS with or without human recombinant Noggin (Regeneron Pharmaceuticals) at 300 ng/ml. Cultures were left to grow for 48 hours then fixed with quick-fix (Culling et al., 1995
) and processed for paraffin wax sectioning.
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RESULTS |
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The addition of the Bmp antagonist noggin to whole eye explants at E10.5 resulted in the development of smaller lenses after 48 hours of culture. We measured the total area (in arbitrary units) of the largest lens section of five noggin-treated and six control explants and showed that there was a statistically significant difference (Fig. 1A). The smaller size of the lenses could also be seen in histological sections of control (Fig. 1B) and noggin-treated (Fig. 1C,D) explants. As primary fiber cells make up most of the area of a lens section at the stage the explant assay was performed, these data indicate that directly or indirectly, Bmps are required for primary fiber cell development. Difficulty in orienting explants of this size precluded a meaningful analysis of primary fiber cell development asymmetries.
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To test the consequences for lens development of Alk6DN expression, we generated transgenic mice with both the EE-1.0-K-Alk6DN and A-Alk6DN constructs. As phenotypes were mild in heterozygous mice, we bred a number of lines to homozygosity. The most severe defects were observed in
A-A6DN line 11 and of EE-A6DN line 48 homozygous animals. Homozygous mice from these two lines were used in all subsequent analysis. For brevity, homozygous mice are referred to by the transgene designation. A summary of the phenotypes in transgenic lines is given in Table 1.
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Dominant-negative Alk6 inhibits primary lens fiber cell differentiation
To permit a detailed assessment of the phenotype in A-Alk6DN and EE-K-Alk6DN transgenic mice, we performed a histological analysis from E9.5-E18.5. No phenotypic change was observed during the induction phases of lens development, but clear defects were observed in primary fiber cell elongation. Lenses of E13.5 mouse embryos from lines of both EE-1.0-K-Alk6DN and
A-Alk6DN transgenics show primary fiber cell elongation defects. Interestingly, however, the defect was not distributed evenly across the lens width. This was most obvious in Hemotoxylin stained sections (Fig. 3A-C) when examining the boundary between equatorial cells and the primary fiber cell mass. In wild-type lenses, this boundary exists only anterior to the equator (Fig. 3A) but in EE-1.0-K-Alk6DN line 48 and
A-Alk6DN line 11 transgenics, the boundary extends close to the posterior lens pole but only on the nasal side (Fig. 3B,C). The nature of the defect is emphasized by the distribution of the fiber cell differentiation markers
-crystallin and MIP26 (Fig. 3D-I). In the E13.0 wild-type lens, both
-crystallin and MIP26 are restricted to the fiber cells and are found at high levels of immunoreactivity along the posterior aspect of the lens from equator to pole (Fig. 3D,G). By contrast, E13.0 lenses from both
A-Alk6DN and EE-1.0-K-Alk6DN transgenics show nasal side domains that have dramatically reduced levels of immunoreactivity for both markers (Fig. 3E-I). This indicates that expression of the dominant-negative Alk6 from either transgene construct inhibits primary fiber cell differentiation.
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In histological sections taken for analysis, transgenic lenses often appeared flatter (a shorter pole-pole distance) than their wild-type littermates. To assess this, we measured the dimensions of E13.5 lenses from wild-type, EE-1.0-K-Alk6DN line 48 homozygous and A-Alk6DN line 11 homozygous embryos. Transgenic lenses of both lines were not significantly smaller equator-to-equator, but the graph of lens shape (Fig. 4) shows that there are significant differences in pole-pole distance. This might be expected in lenses where the primary fiber cell development is perturbed as the elongation of these cells expands the lens along the pole-pole axis.
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Activated phospho-Smads are detected in the primary fiber cell precursors
The Smads are a family of signal transduction molecules active in the Bmp pathways and their phosphorylation indicates that a given cell is responding to Bmp signals (Miyazono et al., 2000). Taking advantage of recently generated antibodies specific for the type 1 receptor phosphorylated forms of Smad1, Smad5 and Smad8 (Korchynskyi et al., 1999
), it was possible to directly examine whether Bmp signaling pathways were functional in the developing lens. This strategy allows the spatial pattern of Bmp responses to be examined. We also chose to use antibodies to the cytoplasmic domain of Bmpr2 (Gilboa et al., 2000
) to assess its distribution. Though Bmpr2 has been reported to be expressed in the adult rat eye (Obata et al., 1999
) the expression pattern in the developing mouse eye has not so far been described.
To further validate the phospho-Smad and Bmpr2 antibodies in the mouse, we performed immunofluorescence labeling of embryonic regions where Bmps were known to be expressed. Specifically, we examined the surface ectoderm in the region of the E9.5 olfactory placode and first branchial arch as in these locations, there is strong expression of Bmp4 (Fig. 6A) (Dudley and Robertson, 1997). With ligand expression, the Bmp signaling pathway might be expected to be active. Anti-phospho-Smad antibody labeling showed strong nuclear immunoreactivity in the epithelium of the olfactory placode and first branchial arch (Fig. 6B,C, red arrowheads) in a pattern that corresponded closely with the domain of Bmp4 expression. The anti-Bmpr2 antibodies also labeled an adjacent section in the olfactory placode and first branchial arch epithelium but the labeling pattern appeared more cell surface than nuclear (Fig. 6D,E, red arrowheads). This indicated a strong spatial correlation between the Bmp4 ligand, the Bmp type 2 receptor and nuclear phospho-Smad labeling suggesting that we are able to detect cells in which this pathway is active.
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DISCUSSION |
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The anti-phospho-Smad1 antibody we have used in this analysis detects phosphorylated Smad1, Smad5 and Smad8 (Korchynskyi et al., 1999). In some circumstances this antibody crossreacts at a low level with phospho-Smad3 (that functions downstream of Tgfß receptors) (Korchynskyi et al., 1999
) potentially complicating interpretation of the current data. Importantly however, there is evidence that at physiological expression levels, Tgfß receptors do not dimerize with Bmp receptors (Massagué, 1998
) and so there is no expectation that dominant-negative receptors from each class would cross-react. Thus, with the current analysis and recent work in which the role of Tgfß signaling in lens development was investigated (de Iongh et al., 2001
) we can conclude that both Bmp and Tgfß signaling are independently important in the development of the primary lens fiber cells. This suggestion is reinforced by the observation that primary fiber cell development can be inhibited in explant culture by noggin (Fig. 1), an inhibitor of BMP but not Tgfß activity.
Bmp signaling in the different phases of lens development
The published evidence indicates that both Bmp7 and Bmp4 are expressed in the eye primordium (see Fig. 7 for summary) and suggests that they have important roles in induction of the lens (Furuta and Hogan, 1998). Bmp7-null mice demonstrate that Bmp7 regulates expression of the transcription factor Pax6 in the lens placode (Wawersik et al., 1999
). As Pax6 is both necessary (Ashery-Padan et al., 2000
) and sufficient (Altmann et al., 1997
) for the earliest stages of lens development, this has argued that Bmp7 has an important role in lens induction (Wawersik et al., 1999
). By contrast, Bmp4 does not regulate expression of Pax6, but is required for the upregulation of Sox2 (Furuta and Hogan, 1998
), a transcription factor that is involved in regulating the expression of crystallin genes sometimes in a complex with Pax6 (Kamachi et al., 2001
). As the ectoderm enhancer used to direct transgene expression in the EE-1.0-K-Alk6DN transgenic mice is expected to be expressed in the lens lineage from E8.75 (Williams et al., 1998
), the question arises of why we do not see a defect in lens induction. There are several reasons. One explanation is that the level of Bmp4 and Bmp7 signaling activity in the induction phases of lens development is high, and that the transgene we describe does not provide a sufficiently high level of expression to inhibit Bmp signaling pathways. Another, more satisfying, explanation is based on the biochemistry of the Bmp receptor signaling system.
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Bmp ligands in the developing eye may be asymmetrically distributed
The data we describe shows that the lens normally goes through a stage where development is anatomically asymmetrical. Specifically, we show that primary lens fiber cells begin to elongate first in the ventrotemporal quadrant of the lens at E12.5, but by E13.5, the nasal side cells have also elongated and the obvious asymmetry is lost. To our knowledge, this is the first time that this aspect of mouse lens development has been recognized.
In the wild-type lens vesicle, Bmpr2 and phospho-Smad immunoreactivity are present in the entire equator and this suggests that all these cells are responding to Bmp signals. This raises the question of why, in the A-Alk6DN and EE-1.0-K-Alk6DN transgenic mice, where lens vesicle stage expression is not expected to be asymmetrical, the primary fiber cell differentiation defect is. The most obvious explanation is that there may be a Bmp ligand that is important for primary fiber cell differentiation that is restricted in its distribution to the ventronasal quadrant of the eye. A preliminary analysis of the expression patterns of many Bmp family members in the primordial eye shows that many are expressed and that the patterns are complex (S. C. F. and R. A. L., unpublished). Given the possibility that Bmps can act as both homodimers and heterodimers and have distinct signaling activity as a consequence (Suzuki et al., 1997
), coupled with the existence of soluble modulators of their activity (Cho and Blitz, 1998
), it is not unreasonable to suggest that Bmp responses within the eye may be finely restricted spatially.
Thus, we propose a model in which the normal lens vesicle contains two subpopulations of primary lens fiber cell progenitors that respond to distinct differentiation stimuli. We propose that in the ventrotemporal and dorsal lens vesicle, there is an early differentiating population (Fig. 8, blue) in which fiber cell elongation is unaffected by dominant-negative Alk6. This could mean that this domain of lens vesicle cells differentiates in response to non-Bmp ligands, or that if a Bmp ligand is involved (as the uniform distribution of phospho-Smad might suggest), it does not bind efficiently to Alk6-containing receptor heterodimers. We also suggest that there is a group of lens vesicle cells located on the nasal side that differentiates later (Fig. 8, red) in response to a Bmp ligand that can be inhibited by dominant-negative Alk6. This model explains both the normal asymmetry in primary fiber cell differentiation and the asymmetrical phenotype of the A-Alk6DN and EE-1.0-K-Alk6DN transgenic mice.
Other analyses of eye development have identified mutant phenotypes and gene expression patterns that reflect asymmetries. Gene targeting of the retinoid receptors Rxra and Rxrg has shown that this pathway is important for development of the ventral eye (Kastner et al., 1994). In particular, compromised function of these receptors resulted in a ventral rotation of the entire lens suggesting that retinoid signaling is important for development of ventral eye structures. It may be interesting to determine whether retinoid signaling and Bmp-mediated primary fiber cell differentiation are developmentally related. Another example of interest is the expression patterns of the forkhead family members Foxg1 and Foxd1. These are expressed in the nasal and temporal retina, respectively (Dirksen and Jamrich, 1995
; Hatini et al., 1994
; Tao and Lai, 1992
), and it is possible that this type of gene expression pattern might reflect spatially restricted fiber cell differentiation stimuli that are likely to have their origin in the developing retina.
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ACKNOWLEDGMENTS |
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