QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Faber, S. C. Articles by Lang, R. A. Search for Related Content PubMed PubMed Citation Articles by Faber, S. C. Articles by Lang, R. A.

Fgf receptor signaling plays a role in lens induction

Sonya C. Faber, Patricia Dimanlig, Helen P. Makarenkova, Sanjay Shirke, Kyung Ko and Richard A. Lang^*,

Skirball Institute for Biomolecular Medicine, Developmental Genetics Program, Cell Biology and Pathology Departments, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
^* Present address: Divisions of Developmental Biology and Ophthalmology, Children’s Hospital Research Foundation, 3333 Burnett Avenue, Cincinnati, OH 45229-3039, USA

View larger version (57K): [in a new window]   Fig. 1. Expression of the lens induction marker Pax6 is dependent on Fgfr kinase activity. (A,B) Mouse half-heads from P6 5.0-lacZ embryos were explanted at E8.5 and cultured for 8 hours either in the absence (A) or presence (B) of 10 µM SU9597. At this stage, X-gal staining reveals only a modest reduction of reporter construct expression in SU9597-treated explants. (C-F) Mouse half-heads from P6 5.0-lacZ embryos were explanted at E8.5 and cultured for 24 hours either in the absence (C,D) or presence (E,F) of 10 µM SU9597. X-gal staining reveals that at this stage, there is a dramatic reduction in reporter construct expression levels in the lens placode in presence of SU9597 whether assessed in whole-mount (compare C with E) or in section (compare D with F, arrowheads). All explants shown in A-F were derived from the same litter of P6 5.0-lacZ embryos. (G-J) Mouse half-heads from wild-type embryos were explanted at E8.5 and cultured for 24 hours either in the absence (G,H) or presence (I,J) of 10 µM SU9597. Frozen sections of the explants were labeled with ant-Pax6 antibodies using indirect immunofluorescence. Both the green and yellow colors show Pax6 immunoreactivity, but the yellow shows the peak signal intensity. This indicates that Pax6 levels are reduced in explants treated with the Fgfr kinase inhibitor. This is true for the lens placode (arrowheads) but is also apparent in the optic vesicle (ov). (K,L) As in G-J, but explants were performed at E9.5 and allowed to proceed for 24 hours. In this case, it is apparent that SU9597 reduces the level of Pax6 immunoreactivity, particularly in the deepest epithelium of the lens pit (lp). The lens pit of the treated explant (L) is also narrower than in the control (K). Explants in A-C,E are shown at the same magnification. All panels are labeled with the approximate stage of development reached at the end of the explant period.

View larger version (64K): [in a new window]   Fig. 2. Tfr transgene construct and expression. (A) Schematic showing the Tfr construct. Segments derived from the mouse Pax6 gene are shown in reds and include the ectoderm enhancer (EE) and the P0 promoter. The promoter is encompassed in a fragment of approximately 1 kb 5' to the start-point of transcription (right-facing arrow). The Fgfr1IIIc cDNA is positioned downstream of the Pax6 gene elements. The various protein-coding domains within the cDNA are include the signal sequence (SS), three imunoglobulin-like domains (Ig1-3), the acidic domain (blue vertical bar), the CAM-homology domain (purple vertical bar) (Doherty and Walsh, 1996) and the transmembrane domain (dark green vertical bar). Included in the construct is the SV40 virus small t antigen intron and polyadenylation signals. (B-E) Whole-mount in situ hybridization on mouse embryos. (B) Control hybridization with an antisense probe to A-crystallin showing signal in the lens pit in an E10.5, wild-type embryo (1ba – first branchial arch). (C,D) Hybridization of an antisense SV40 probe to Tfr7 embryos at E10.5 (C) and E11.5 (D). Hybridization signal is apparent in the lens pit at E10.5 and the lens vesicle at E11.5. (E) Hybridization of an antisense SV40 probe to a Tfr3 embryo at E12.0 showing signal in the lens epithelium. The broken line in D,E outlines the optic cup. (F) RT-PCR transgene expression analysis on lens placode and optic vesicle (E9.5) from Tfr7 hemizygous mice. Bacteriophage X174 DNA digested with HaeIII is run as a size standard on both sides of the gel (X). The GAPDH cDNA-derived product appears at 420 bp (blue arrowhead). The PCR product derived from the transgene appears at 200 bp (red arrowhead). This analysis shows that reverse-transcriptase (RT) must be included if either PCR product is to be amplified and that the transgene-specific product can be amplified only from lens placode cDNA.

View larger version (101K): [in a new window]   Fig. 3. Histological analysis of Tfr7 mice. At E9.75 in wild-type embryos (A), the lens placode (lpl) has thickened adjacent to the presumptive retina (pr), and the optic vesicle (ov) has begun invaginating. By contrast, Tfr7/Tfr7 embryos derived from the same litters show poorly developed lens placodes and a delay in the invagination of the optic vesicle. Wild-type (C) and Tfr7/Tfr7 (D) eyes at E10.5 showing the arrangement of the lens pit (lp) presumptive retina, presumptive pigmented epithelium (prpe) and periorbital mesenchyme (mc). At this stage, the Tfr7/Tfr7 lens pit is consistently smaller than that in the wild type. Wild-type (E) and Tfr7/Tfr7 (F) mouse eyes at E11.5, showing that the lens vesicle (lv) in Tfr7/Tfr7 embryos is smaller than in wild type and remains attached to the surface ectoderm (red arrowhead). Periorbital mesenchymal cells have been unable to migrate across the full width of the presumptive cornea. At E12.5 in wild-type mice (G) the primary fiber cells (pfc) have extended anteriorly to the lens epithelium (ep). By contrast, in Tfr7/Tfr7 mice (H), no fiber cell differentiation has occurred as indicated by the small, hollow lens vesicle (lv). The lens vesicle remains attached (red arrowheads) to the surface ectoderm (se) and prevents the complete migration of periorbital mesenchyme (arrows). At the day of birth, both wild-type (I) and Tfr7/Tfr7 (J) lenses have developed both primary and secondary fiber cells (pfc and sfc, respectively) but the Tfr7/Tfr7 lens is significantly smaller. There is also a persistent lens stalk in the transgenic (red arrowhead). Whole-mount preparations of wild-type (K) and Tfr7/Tfr7 (L) eyes also indicate the existence of the persistent lens stalk (red arrowhead). al, anterior lens; cor, cornea; id, iris diaphragm.

View larger version (58K): [in a new window]   Fig. 4. Proliferation levels in the Tfr3 and Tfr7 lens lineage. (A) Histological section from an E10.5 mouse embryo showing typical BrdU labeling; the blue fluorescence identifies all nuclei, while the red identifies BrdU-positive cells. Both the total number of cells and the number of BrdU-positive cells in the lens pit were counted within the boundaries indicated (white lines) and the proportion of positive cells calculated. The results of this analysis for wild-type and Tfr7/Tfr7 homozygous mice is shown in B. This indicates that there is a statistically significant reduction in proliferative index in the transgenics at E10.5. (C) An E13.5 wild-type lens section showing typical detection of BrdU incorporation. The section is overlaid with the radial grid used for counting. The graph (D) shows the BrdU labeling index at E13.5 in a series of radial sectors that represent different lens domains. In wild-type mice (blue trace) there are two peaks of proliferation at 20° and 160°; this represents a location adjacent to the immature anterior chamber and is a typical proliferation pattern for the mouse lens (McAvoy, 1978). The Tfr3/Tfr3 (orange trace) and Tfr7/Tfr7 (red trace) transgenic mice show reduced levels of proliferation; the Tfr7/Tfr7 transgenic mice are more severely affected. Standard errors are represented by vertical bars.

View larger version (130K): [in a new window]   Fig. 5. Fgf receptor and Bmp7 signaling cooperate in lens development. All panels show Hematoxylin and Eosin stained sections of P0 mouse eyes. (A) Wild-type eye. (B) Tfr7/Tfr7 mouse eye with persistent lens stalk (red arrowhead). The blue arrow indicates the eyelid suture. (C,D) Bmp7-/- eyes showing the range of possible phenotypes manifesting as microphthalmia (C) and anophthalmia (D). Only the eyelid suture (blue arrow) is recognizable in the case of anophthalmia (D is twice the magnification of other panels). This can be compared with the eyelid suture in B (blue arrow). (E) Eye section from Tfr7/Tfr7, Bmp7+/- mouse showing the typical phenotype featuring failure of lens vesicle closure and separation (red arrowhead), and extrusion of eosinophilic fiber cell material into the conjunctival sac (black arrows). (F) Eye section from a second Tfr7/Tfr7, Bmp7+/- mouse showing failure of lens vesicle closure and separation (red arrowhead) and extrusion of fiber cell material into the vitreous (arrowheads). (G,H) Two different examples of eye sections from Tfr7/Tfr7, Bmp7-/- mouse eyes showing the disproportionate effects on lens development compared with Bmp7-/- mouse eyes (C). In these cases, the lens (arrowheads) is disrupted (G) and smaller than observed in any other genotype (G,H).

View larger version (100K): [in a new window]   Fig. 6. Pax6, Foxe3 and Sox2 are downregulated in Tfr7/Tfr7, Bmp7+/- mice. Immunohistochemical detection of Pax6 in the developing eyes of wild-type (A,C) Tfr7/Tfr7 (B,D) and Tfr7/Tfr7, Bmp7+/- (E) embryos at E9.5. In all cases, the lens placode is indicated by the arrowheads. Both the green and yellow colors show Pax6 immunoreactivity, but the yellow shows the peak signal intensity. A,B represent one experiment and indicate that there is a reduction in the level of Pax6 immunoreactivity in the lens placode of Tfr7/Tfr7 mice. The second experiment shows the lens placode at higher magnification and indicates that the level of Pax6 immunoreactivity is progressively reduced in Tfr7/Tfr7 (D) and Tfr7/Tfr7, Bmp7+/- (E) embryos. (F-H) Immunohistochemical detection of Pax6 in wild-type (F) Tfr7/Tfr7 (G) and Tfr7/Tfr7, Bmp7+/- (H) embryos at E10.5. The lens pit (lp) epithelium is demarcated by white arrowheads. pr, presumptive retina; rpe, presumptive retinal pigmented epithelium. These panels show that the lens pit is smaller and the per-cell Pax6 immunoreactivity lower in Tfr7/Tfr7 and Tfr7/Tfr7, Bmp7+/- mice. For experiments 1-3, Pax6 immunoreactivity in the presumptive retina (pr) is also reduced in Tfr7/Tfr7 (B,D,G) and Tfr7/Tfr7, Bmp7+/- (E,H) embryos compared with wild-type (A,C,F). (I-K) Whole-mount in situ hybridization with an antisense probe to Foxe3 in wild-type (I) Tfr7/Tfr7 (J) and Tfr7/Tfr7, Bmp7+/- (K) mice at E10.5. This shows that the level of Foxe3 expression is reduced in Tfr7/Tfr7, Bmp7+/- mice. The first branchial arch (1ba) is outlined in gray and the optic cup with a broken black line. (L-N) Thick sections of embryos hybridized with an antisense probe to Sox2 in wild-type (L) Tfr7/Tfr7 (M) and Tfr7/Tfr7, Bmp7+/- (N) mice at E10.5. This shows that Sox2 expression in the lens pit (lp and red arrowheads) cannot be detected in Tfr7/Tfr7 or Tfr7/Tfr7, Bmp7+/- mice.

View larger version (14K): [in a new window]   Fig. 7. A model for the genetic pathway describing lens induction and development. The arrows indicate genetic interactions determined by these and previous analyses. Gray arrows indicate a possible rather than a demonstrated pathway element. At the apex of the hierarchy is the pre-placodal phase of Pax6 expression (Pax6pre-placode). It is understood that the later phase of Pax6 expression in the lens placode (Pax6placode) is dependent upon earlier function of Pax6. The genetic pathway described reflects this interaction. As we observe that Fgfr and Bmp7 signaling cooperate to maintain the placodal phase of Pax6 expression, it follows that their input to the pathway must be upstream of Pax6placode. Previous analysis has shown that the early phase of Pax6 expression is unaffected in the Bmp7-null mice and, thus, Fgfr and Bmp7 signaling must converge on the pathway downstream of Pax6pre-placode. Evidence that Foxe3 is downstream of Pax6placode and Fgfr and Bmp7 signaling includes (1) the reduced level of Foxe3 expression in Tfr7/Tfr7, Bmp7+/- embryos, (2) similar phenotypes in Tfr7/Tfr7, Bmp7+/- and dyl (Foxe3dyl/dyl) embryos, (3) the absence of Foxe3 expression in small eye embryos, and (4) the absence of Foxe3 expression in embryos carrying a targeted deletion of the Pax6 ectoderm enhancer (Dimanlig et al., 2001). Thus, Foxe3 expression must lie upstream of events such as lens lineage proliferation, vesicle closure and separation. While we currently do not understand the genetic relationship between Foxe3 and Sox2, it is clear from this and previous analyses performed that Sox2 lies downstream of Pax6placode. Because Sox2 (but not Pax6) expression is diminished in the Bmp4-null mice, Bmp4 signaling must contribute to the pathway between Pax6placode and Sox2.