Intracellular distribution of type II collagen mRNA and prolyl 4-hydroxylase in embryonic avian corneal epithelia

Abstract

The distribution of endoplasmic reticulum (ER) proteins and type II collagen mRNA in whole mount preparations of embryonic corneal epithelia was investigated. We asked, do transmission electron microscopy (TEM) and confocal microscopy of chick prolyl 4-hydroxylase (CPH) show similar ER distributions as CPH is necessary for collagen biosynthesis? The overall distribution of ER was analyzed by TEM. Collagen secreting ER was localized with antibodies to chick prolyl 4-hydroxylase (CPH, CPH-alpha, or CPH-beta subunits). Type II collagen mRNA distribution was determined by in situ hybridization. The ER localized with TEM, and immunohistochemistry using anti-CPH antibodies, showed similar distribution patterns. Immunostaining for CPH and CPH-beta was prominent in both periderm and basal cells and also appeared to stain the periderm apical surface. CPH-alpha was less intense, only localizing to ER regions. The basal cell CPH distribution appeared perinuclear in optical sections that contained nuclei, but occupied nearly all the cytoplasm in a reticular pattern above and below nuclei, similar to that seen with TEM cross-sections. Epithelia double labeled with propidium iodide and CPH showed overlapping cytoplasmic staining. The distribution of type II collagen mRNA was similar to the ER staining pattern, appearing to represent a subset of total ER. This study demonstrates that ER markers have a similar distribution as type II collagen mRNA in embryonic avian corneal epithelia. In addition the CPH subunits had distinct, but overlapping distributions, suggesting that they may act independently.