Keratan sulfate glycosaminoglycan and the association with collagen fibrils in rudimentary lamellae in the developing avian cornea.
OBJECTIVE
Keratan sulfate (KS), through its association with fibrillar collagen as KS-substituted proteoglycan (KS PG), is thought to be instrumental in the structural development of the corneal stroma. The authors used two different sulfate motif-specific antibodies to identify the sequence of appearance, and the association with collagen, of sulfated KS during avian corneal morphogenesis.


METHODS
Corneas from chicken embryos throughout the developmental period, from day 8 through day 18 of incubation, were examined by immunofluorescence and immunoelectron microscopy using monoclonal antibodies 5D4 and 1B4, which react with high- and low-sulfated epitopes on KS, respectively.


RESULTS
KS was identified as punctate labeling at incubation day 8, the earliest stage examined, suggesting a cell-associated distribution. By day 10, labeling was more homogeneous, indicating that KS sulfation motifs were present in the stromal extracellular matrix. At day 12 through day 14, immunopositive sites were concentrated primarily in the anterior stroma but became more uniform throughout the full stromal thickness by day 18. From day 10 on, electron microscopy revealed a high-sulfated KS epitope closely associated with bundles of regularly arranged collagen fibrils, initially near cell surfaces in rudimentary lamellae. Individual cells, associated with collagen bundles with different fibril orientations, imply the potential for simultaneous deposition of multiple lamellae.


CONCLUSIONS
During chick corneal morphogenesis, significant matrix deposition of high-sulfated KS epitope occurs by day 10, with accumulation subsequently proceeding in an anterior-to-posterior manner. High-sulfated KS likely serves to help define the regular spatial organization of collagen fibrils in bundles newly extruded into the extracellular milieu.
Young
Robert D
RD
Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom.
Gealy
E Claire
EC
Liles
Melody
M
Caterson
Bruce
B
Ralphs
James R
JR
Quantock
Andrew J
AJ
D000818:Animals; D002642:Chick Embryo; D003315:Cornea / Q000196:embryology* / Q000378:metabolism* / Q000648:ultrastructure; D003319:Corneal Stroma / Q000196:embryology / Q000378:metabolism / Q000648:ultrastructure; D024022:Fibrillar Collagens / Q000378:metabolism* / Q000648:ultrastructure; D007632:Keratan Sulfate / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009024:Morphogenesis
D024022:Fibrillar Collagens; D007632:Keratan Sulfate