Spry1 and Spry2 are necessary for eyelid closure.
Sproutys (Sprys) are downstream targets and negative feedback regulators of the FGF-Ras-ERK signaling pathway. Our previous studies have shown that Spry1 and Spry2, through negative modulation of FGF-ERK signaling, allow lens vesicle separation from the overlying ectoderm and regulate corneal epithelial proliferation. Here we show that Spry1 and Spry2 are necessary for eyelid closure. Murine palpebral conjunctival epithelial cells that differentiate as inner eyelids and adjacent mesenchymal cells express Spry1 and Spry2 prior to eyelid closure. Conditional deletion of both Spry1 and Spry2, but not either one alone, in the ocular surface epithelial cells result in the "EOB" (eyes open at birth) phenotype suggesting redundant roles for these proteins during eyelid closure. Spry mutant eyelids show increased proliferation of conjunctival epithelial cells with concomitant induction of FGF targets, Erm, Pea3 and Dusp6 and elevated ERK phosphorylation. Peridermal cells at the leading edge of Spry-mutant eyelids showed reduced c-Jun, but not ERK, phosphorylation, reduced F-actin polymerization and reduced motility in vitro. Spry mutant eyelids also showed disruptions in epithelial mesenchymal interactions reflected in the enhanced mesenchymal Spry1 and Spry4 expression, disaggregation of BMP4-positive mesenchymal cells and loss of Shh in the eyelid epithelium. Spry mutant eyelids also showed increased Wnt signaling and reduced expression of Foxc1 and Foxc2, two transcription factors previously shown to be necessary for eyelid closure. Collectively, our results show that conjunctival epithelial Spry1 and Spry2 redundantly promote eyelid closure by (a) stimulating ERK-independent, c-Jun-mediated peridermal migration, (b) suppressing conjunctival epithelial proliferation through FGF-ERK signaling, (c) mediating conjunctival epithelial-mesenchymal interactions and (d) maintaining expression of Foxc1 and Foxc2.
Department of Surgery, 253 Criss III, Cancer Center, 2500 California Plaza, Creighton University, Omaha, NE 68178, USA.
Murali R
MR
Kuracha
Ed
E
Siefker
Jonathan D
JD
Licht
Venkatesh
V
Govindarajan
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D002465:Cell Movement; D049109:Cell Proliferation; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D000078404:Epidermal Cells; D004848:Epithelium / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005143:Eyelids / Q000166:cytology / Q000196:embryology / Q000201:enzymology / Q000502:physiology*; D005346:Fibroblast Growth Factors / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D053823:Hedgehog Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D060449:Wnt Signaling Pathway
D048868:Adaptor Proteins, Signal Transducing; D055415:Bone Morphogenetic Protein 4; D051858:Forkhead Transcription Factors; C112890:Foxc1 protein, mouse; D053823:Hedgehog Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D016755:Proto-Oncogene Proteins c-jun; C465570:Shh protein, mouse; C425468:Spry1 protein, mouse; C499037:Spry4 protein, mouse; C082078:mesenchyme fork head 1 protein; D005346:Fibroblast Growth Factors; D017346:Protein Serine-Threonine Kinases; C118389:Spry2 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases
C-Jun; ERK; Eyelids; FGF; Foxc1; Foxc2; Sprouty; Wnt