To assess alterations in lens and corneal development in
Spry-null mice, sections of embryos that had lost two, three, or all four alleles of
Spry1 and -
2 in the lens, corneal, and conjunctival epithelial cells were analyzed by histology (
Fig. 4). Embryos that lacked Cre recombinase (Cre−) were used as controls. At E10.5, embryos lacking two alleles of
Spry2 (
Fig. 4B), one allele of
Spry1 and two alleles of
Spry2 (
Fig. 4C), and all four alleles of
Spry1 and -
2 (
Fig. 4D) were indistinguishable from control embryos (
Fig. 4 A). In these mutants, the lens pit formed normally. At E11.5, a remnant of a lens stalk, a transient structure connecting the lens vesicle to the presumptive cornea, was seen in control embryos (
Figs. 4E,
4E′, arrow).
Spry mutants, in contrast, displayed a thicker stalk with more cells (
Figs. 4F–H,
4F′–4H′, arrows). At E12.5, the lens vesicle in the control embryo had separated from the presumptive corneal epithelium, and periocular mesenchymal cells were present in the region between the lens and the presumptive cornea (
Figs. 4I,
4I′). In contrast,
Spry mutant embryos still contained the lens stalk and failed to separate from the overlying corneal epithelium (
Figs. 4J–L,
4J′–L′, arrows). The stalks in the Spry mutant embryos were persistent at later ages including E15.5 (
Figs. 4N–P,
4N′–P′). The posterior part of the stalk was continuous with the lens and the anterior part with the corneal epithelium (
Figs. 4N′–P′). Occasionally, cells within the stalk piled up (
Figs. 4P,
4P′) and lenses were ruptured in the
Spry mutant embryos (
Figs. 6L,
8F,
9E). Fiber cells in the posterior portion of the lens in
Spry mutants (
Figs. 4J–L,
4N–P) appeared similar to those in control embryos (
Figs. 4I,
4M). The mean lens diameter was not significantly altered in
Spry mutants compared with Cre− controls at E12.5 (0.208 ± 0.022 mm [
Spry1;
Spry2 double mutants] vs. 0.191 ± 0.014 mm [
Cre−]) or at E15.5 (0.490 ± 0.019 mm [
Spry1;
Spry2 double mutants] vs. 0.480 ± 0.011 mm [
Cre−]). The corneal stroma and endothelium in
Spry mutants (in the posterior part of the cornea) were discontinuous, presumably due to the presence of the lens stalk (
Figs. 4N–P,
4N′–P′). Embryos that lacked two alleles of
Spry1 (
Supplementary Figs. S1A–B′) or one allele each of
Spry1 and -
2 (
Supplementary Figs. S1A–B′) also displayed lens stalks and were identical with other
Spry mutants. As loss of two, three, or four alleles of
Spry1 and/or -
2 leads to similar alterations in early lens differentiation, detailed analysis was performed on embryos that lacked all four alleles of
Spry1 and -
2 (unless specified otherwise). In this article, we report changes in lens and corneal differentiation. Alterations in ocular gland development and eyelid closure were also seen but are not reported here.