The negative regulatory Spred1 and Spred2 proteins are required for lens and eye morphogenesis.

The transparent and refractive properties of the ocular lens are dependent on its precise cellular structure, supported by the regulation of lens cellular processes of proliferation and differentiation that are essential throughout life. The ERK/MAPK-signalling pathway plays a crucial role in regulating lens cell proliferation and differentiation, and in turn is regulated by inhibitory molecules including the Spred family of proteins to modulate and attenuate the impact of growth factor stimulation. Given Spreds are strongly and distinctly expressed in lens, along with their established inhibitory role in a range of different tissues, we investigated the role these antagonists play in regulating lens cell proliferation and differentiation, and their contribution to lens structure and growth. Using established mice lines deficient for either or both Spred 1 and Spred 2, we demonstrate their role in regulating lens development by negatively regulating ERK1/2 activity. Mice deficient for both Spred 1 and Spred 2 have impaired lens and eye development, displaying irregular lens epithelial and fibre cell activity as a result of increased levels of phosphorylated ERK1/2. While Spred 1 and Spred 2 do not appear to be necessary for induction and early stages of lens morphogenesis (prior to E11.5), nor for the formation of the primary fibre cells, they are required for the continuous embryonic growth and differentiation of the lens.

Spred negatively regulates lens growth by modulating epithelial cell proliferation and fiber differentiation.

Spred, like Sprouty (Spry) and also Sef proteins, have been identified as important regulators of receptor tyrosine kinase (RTK)-mediated MAPK/ERK-signaling in various developmental systems, controlling cellular processes such as proliferation, migration and differentiation. Spreds are widely expressed during early embryogenesis, and in the eye lens, become more localised in the lens epithelium with later development, overlapping with other antagonists including Spry. Given the synexpression of Spreds and Spry in lens, in order to gain a better understanding of their specific roles in regulating growth factor mediated-signaling and cell behavior, we established and characterised lines of transgenic mice overexpressing Spred1 or Spred2, specifically in the lens. This overexpression of Spreds resulted in a small lens phenotype during ocular morphogenesis, retarding its growth by compromising epithelial cell proliferation and fiber differentiation. These in situ findings were shown to be dependent on the ability of Spreds to suppress MAPK-signaling, in particular FGF-induced ERK1/2-signaling in lens cells. This was validated in vitro using lens epithelial explants, that highlighted the overlapping role of Spreds with Spry2, but not Spry1. This study provides insights into the putative function of Spreds and Spry in situ, some overlapping and some distinct, and their importance in regulating lens cell proliferation and fiber differentiation contributing to lens and eye growth.

Crim1 is required for maintenance of the ocular lens epithelium.

The development and growth of the vertebrate ocular lens is dependent on the regulated proliferation of an anterior monolayer of epithelial cells, and their subsequent differentiation into elongate fiber cells. The growth factor rich ocular media that bathes the lens mediates these cellular processes, and their respective intracellular signaling pathways are in turn regulated to ensure that the proper lens architecture is maintained. Recent studies have proposed that Cysteine Rich Motor Neuron 1 (Crim1), a transmembrane protein involved in organogenesis of many tissues, might influence cell adhesion, polarity and proliferation in the lens by regulating integrin-signaling. Here, we characterise the lens and eyes of the Crim1KST264 mutant mice, and show that the loss of Crim1 function in the ocular tissues results in inappropriate differentiation of the lens epithelium into fiber cells. Furthermore, restoration of Crim1 levels in just the lens tissue of Crim1KST264 mice is sufficient to ameliorate most of the dysgenesis observed in the mutant animals. Based on our findings, we propose that tight regulation of Crim1 activity is required for maintenance of the lens epithelium, and its depletion leads to ectopic differentiation into fiber cells, dramatically altering lens structure and ultimately leading to microphthalmia and aphakia.

Conditional Ablation of Spred1 and Spred2 in the Eye Lens Negatively Impacts Its Development and Growth.

The development and growth of the eye depends on normal lens morphogenesis and its growth. This growth, in turn, is dependent on coordinated proliferation of the lens epithelial cells and their subsequent differentiation into fiber cells. These cellular processes are tightly regulated to maintain the precise cellular structure and size of the lens, critical for its transparency and refractive properties. Growth factor-mediated MAPK signaling driven by ERK1/2 has been reported as essential for regulating cellular processes of the lens, with ERK1/2 signaling tightly regulated by endogenous antagonists, including members of the Sprouty and related Spred families. Our previous studies have demonstrated the importance of both these inhibitory molecules in lens and eye development. In this study, we build on these findings to highlight the importance of Spreds in regulating early lens morphogenesis by modulating ERK1/2-mediated lens epithelial cell proliferation and fiber differentiation. Conditional loss of both Spred1 and Spred2 in early lens morphogenesis results in elevated ERK1/2 phosphorylation, hyperproliferation of lens epithelia, and an associated increase in the rate of fiber differentiation. This results in transient microphakia and microphthalmia, which disappears, owing potentially to compensatory Sprouty expression. Our data support an important temporal role for Spreds in the early stages of lens morphogenesis and highlight how negative regulation of ERK1/2 signaling is critical for maintaining lens proliferation and fiber differentiation in situ throughout life.