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Bmp signaling is required for development of primary lens fiber cells

¹ Division of Developmental Biology and Department of Ophthalmology, Children’s Hospital Research Foundation, 3333 Burnet Avenue Cincinnati, OH 45229, USA
² Division of Human and Molecular Genetics, Children’s Research Institute, 700 Children’s Drive, Columbus, OH 43205, USA
³ The Neurosciences Institute, 10640 John Jay Hopkins Drive, San Diego, CA 92121, USA

View larger version (142K): [in a new window]   Fig. 1. Noggin-treated lens explants have reduced development of primary lens fiber cells. E10.5 mouse eye primordia were explanted into collagen gel in culture either in the absence [A (red bar) and B] or presence [A (blue bar), C,D] of the Bmp inhibitor noggin at 300 ng/ml. Quantification of the area of the largest lens section (in a series) in control and experimental explants indicated that noggin treatment reduced lens growth (A). At this stage of development, primary lens fiber cells make up most of the area in a lens section. The larger size and improved development of primary lens fiber cells in untreated explants (B) can be observed by comparison with those that were noggin-treated (C,D).

View larger version (56K): [in a new window]   Fig. 2. Alk6DN transgene constructs and lens expression. (A) The EE-1.0-K-Alk6DN and A-Alk6DN transgene constructs. The positions of the Pax6 ectoderm enhancer (EE, blue box) and the Pax6 P0 promoter region (P6 1.0, green box) in EE-1.0-K-Alk6DN are shown. In A-Alk6DN the promoter region is indicated by the purple box. In both constructs, the dominant-negative Alk6-coding region (containing a point mutation giving K231R) is indicated in yellow. Included in both constructs is the SV40 virus small t antigen gene region (gray) that contains splicing and polyadenylation signals. In the EE-1.0-K-Alk6DN construct, the translation start codon was engineered to the most efficient consensus sequence as defined by Kozak (Kozak, 1986). (B) Schematic (not to scale) of the expression pattern of the EE containing promoters (B top, blue) and the A promoter (B bottom, purple). The EE promoter begins expression at E9.5 in the surface ectoderm, continues to be expressed at E10.5 in the lens pit. By E11.5 and through E13.5 it is expressed in all of the cells of the lens, but in adulthood, expression is restricted to the lens epithelial layer. Expression of transgenes driven by the A promoter begins at E11.0 in differentiating primary fiber cells and continues in all lens fiber cells into adulthood. (C-H) Whole-mount in situ hybridization. Hybridization of an antisense SV40 probe to a positive control, the P6 5.0-lacz reporter line at E13.5 is shown in C. Two negative controls for the E13.5 lens, follistatin (D) and noggin (E) are shown adjacent. Follistatin labels nasal periocular mesenchyme at E13.5. An SV40 probe hybridization signal is also observed in the lens of three separate lines of transgenic mice including A-Alk6DN-88 at E13.5 (F) A-Alk6DN-11 at E11.5 (G) and EE-1.0-K-Alk6DN-48 at E11.5 (H). (I-K) Matched brightfield (upper panel) and darkfield (lower panel) of section in situ hybridization with radioactively labeled antisense SV40 probe on E12.5 eye tissue from (I) EE-1.0-K-Alk6DN-48, (J) A-Alk6DN-88 and (K) wild-type control.

View larger version (129K): [in a new window]   Fig. 3. Histological analysis of A-Alk6DN and EE-1.0-K-Alk6DN transgenic lenses. E13.5 Hematoxylin and Eosin stained sections of wild-type mouse eyes (A), and homozygous mice of the A-Alk6DN-11 (B) and EE-1.0-K-Alk6DN-48 (C) transgenic lines. The black arrowheads indicate the normal nasal side equatorial structure in wild-type mice (A) and the abnormal form (B,C) seen with both transgenic mouse constructs. (D-F) -crystallin immunolabeling of E13.0 lenses from wild-type mice (D) and from homozygous A-Alk6DN-11 (E) and EE-1.0-K-Alk6DN-48 (F) transgenics. The white arrowheads indicate the nasal side domain in which primary fiber cells have low -crystallin levels. Similarly, MIP26 immunolabeling in wild-type E13.0 mouse lenses extends to the equator (G), while in homozygous A-Alk6DN-11 (H) and EE-1.0-K-Alk6DN-48 (I) transgenics of the same age, there is a nasal side region in which labeling is absent or low. Sections in A-I are located in the ventral half of the lenses shown. (J-L) Day-of-birth Hematoxylin and Eosin stained sections of the eyes of wild-type mice (J), and homozygous mice of the A-Alk6DN-11 (K) and EE-1.0-K-Alk6DN-48 (L) transgenic lines. This shows that the transgenic lenses are smaller and have emphasized suture spaces (J-L, arrowheads) owing to the lack of complete elongation of the primary fiber cells. (M-O) A comparison of whole-mount lenses dissected from wild-type (M) and homozygous A-Alk6DN-11 (N) and EE-1.0-K-Alk6DN-48 (O) mice showing the refractile anomaly present in the transgenics (N,O, red arrowheads).

View larger version (21K): [in a new window]   Fig. 4. A-Alk6DN and EE-1.0-K-Alk6DN transgenic lenses have a reduced pole-pole dimension. Graph showing a linear regression analysis of the relationship between equator-equator (nasotemporal) and pole-pole dimensions in lens sections taken from wild-type (blue annotation), homozygous A-Alk6DN (orange annotation) and EE-1.0-K-Alk6DN (red annotation) transgenic mice. Measurements taken for this analysis were made on the largest coronal lens sections in a given series. The average dimension for each data set is projected to the axes and marked by the colored dot. The equator-equator dimension in wild-type and transgenic mice is minimally different, but the pole-pole dimension between wild-type and either transgenic is. This indicates that the transgenic lenses are more ellipsoid in shape and that primary fiber cell differentiation is reduced.

View larger version (110K): [in a new window]   Fig. 5. Asymmetry in normal lens development and in the phenotype of Alk6DN transgenic mice. (A-C) Hematoxylin and Eosin stained sections through the ventral eye region of the right eye of an E12.5 wild-type mouse. These are equally spaced serial sections located at 40 µm (A), 80 µm (B) 120 µm (C) from the first ventral lens section. The reference proximodistal axis from the future optic nerve head to the mid point of the developing cornea is marked by the red line in C. These sections show that primary fiber cell elongation begins in a temporally located (T) domain of the posterior wall of the lens vesicle. The nasal (N) aspect of the posterior lens vesicle wall remains undifferentiated at this stage. The relative size of the undifferentiated domains is marked by the broken red lines. It is also observed that there is a larger domain of developing retina on the temporal side of the optic stalk. (D-F) Sections through the ventral eye region of the left eye of the same E12.5 wild-type mouse shown in A-C labeled for the differentiation marker MIP26 (red) and nuclei (green). These are also equally spaced serial sections located at 40 µm (D), 80 µm (E) and 120 µm (F) from the first ventral lens section and serve to emphasize the temporal side (T) primary fiber cell differentiation asymmetry. The relative size of the undifferentiated domains is marked by the broken white lines in F. (G,H) Three dimensional renderings of E13.5 homozygous A-Alk6DN-11 and wild-type lenses in posterior pole (G) and nasal (H) views. The renderings were reconstructed from serial sections. The glassy sphere represents the lens capsule and the purple surface the boundary between epithelium and fiber cell mass. These renderings indicate clearly the ventronasal location of the primary fiber cell differentiation defect in the A-Alk6DN-11 homozygous transgenic lenses.

View larger version (88K): [in a new window]   Fig. 6. Anti-Phospho-Smad and Bmpr2 labeling reveals Bmp responsive cells. (A) Schematic showing the pattern of Bmp4 expression in olfactory placode (OP) first branchial arch (1BA) and otic vesicle (OV) of an E9.5 mouse embryo (Dudley and Robertson, 1997). The approximate region of tissue shown in B-E is indicated by the red boxes. (B) Anti-phospho-Smad labeling (green) of E9.5 mouse section in the region of the first branchial arch (1BA) and olfactory placode (OP). Intense labeling is observed in the epithelia of both structures. A merge of (B) with Hoechst nuclear labeling (blue) is shown in C. The turquoise color of nuclei in (C, red arrowheads) indicates nuclear phospho-Smad immunoreactivity. (D) Bmpr2 immunoreactivity in sections of E9.5 mouse first branchial arch (1BA) and olfactory placode (OP). Cell-surface labeling is apparent in the cells of the epithelia (red arrowheads) and more faintly in the underlying mesenchyme. A merge of (D) with Hoechst labeling (E) shows that, as would be expected for cell-surface labeling, the nuclei do not change color. (F) Anti-phospho-Smad labeling (green) of E12.5 mouse eye sections showing the lens vesicle (LV) and anterior optic cup rim (broken red line) on the nasal side (N). A nuclear pattern of labeling is observed in lens cells at the equator (red arrowheads) faintly in the more posterior presumptive primary lens fiber cells (white arrowheads) and in the optic cup rim. A merge of F with Hoechst nuclear labeling (G, blue) gives turquoise color nuclei thus confirming nuclear phospho-Smad labeling. (H) Bmpr2 immunoreactivity in sections of E11.5 mouse eye. Cell-surface labeling is apparent in the cells at the lens vesicle (LV) equator (red arrowheads) and in the cells of the developing retina and presumptive RPE. A merge of (H) with Hoechst labeling (I) shows that, as would be expected for cell-surface labeling, the nuclei do not change color.

View larger version (53K): [in a new window]   Fig. 7. Summary of the expression patterns of Bmp family ligands and receptors in the developing eye. The different stages of eye development (labelled underneath) are represented by the different line drawings. Dorsal is upwards. The distribution and approximate intensity of labeling for the different ligands and receptors is indicated by the colored stippling. The expression patterns of Bmp4, Bmp7, Alk3 and Alk6 are derived from in situ hybridization experiments presented in a prior publication (Furuta and Hogan, 1998). The distribution of Alk2 is based on antibody labeling (Yoshikawa et al., 2000) and our understanding of Bmpr2 distribution on the current analysis.

View larger version (75K): [in a new window]   Fig. 8. Model for the asymmetrical distribution of primary fiber cell differentiation stimuli in the developing eye. In this model, we propose that there are two distinct stimuli that direct primary fiber cell differentiation. In the ventronasal quadrant of the developing eye, we suggest, based on the phenotype observed in the A-Alk6DN and EE-1.0-K-Alk6DN transgenic mice, that there is a Bmp ligand that can be inhibited by Alk6DN. We suggest that there must be other primary fiber cell differentiation stimuli that are not inhibited by Alk6DN.