Abstract
Purpose :
The lens resides in a hypoxic environment resulting in a diminishing oxygen gradient from surface to core resulting in a hypoxic microenvironment in the microregion of the lens where newly made lens fiber cells initiate their remodeling program to eliminate their organelles and express key fiber cell proteins. This suggests that the hypoxic environment of the lens itself may be required for the development of mature lens structure and transparency. To test this hypothesis, we sought to establish a requirement for hypoxia and key hypoxia-dependent transcriptional regulatory pathways for elimination of organelles and expression of critical proteins during mature lens fiber cell formation.
Methods :
Embryonic chick lenses were cultured under normoxia (21% oxygen) or hypoxia (1% oxygen) for 4 hrs to 5 days in the presence or absence of specific activators (DMOG) or inhibitors (chemotin) of the master regulator of the hypoxic response, hypoxic response hypoxia-inducible factor 1 alpha (HIF1a). Lenses were microdissecrted and elimination of mitochondria (MT), endoplasmic reticulum (ER) and Golgi apparatus (GA) was monitored by quantitative western blotting. These studies were paralleled in primary human embryonic lens cells treated identically and monitored by high-resolution confocal microscopy. High-throughput RNA sequencing was used to identify genome-wide lens fiber cell genes whose expression was dependent on hypoxia and activation of HIF1a. Binding of HIF1a to the promoters of identified genes was monitored by chromatin immunoprecipitation (CHIP) and CUT&RUN analysis followed by high-throughput sequencing and genome-wide mapping.
Results :
Hypoxia and activation of HIF1a are required for lens fiber cell elimination of MT, ER and GA to form mature lens fiber cells composing the lens organelle-free zone. Expression of multiple critical lens fiber cell structural and regulatory genes is dependent on the hypoxic microenvironment of lens fiber cells and activation of the HIF1a-dependent transcriptional regulatory pathway.
Conclusions :
The hypoxic environment of the lens itself is required to attain its mature form and function. Hypoxia-dependent transcriptional regulation by HIF1a drives the elimination of non-nuclear organelles and expression of key lens fiber cell proteins during lens fiber cell remodeling to form mature transparent lens fiber cells.
This is a 2020 ARVO Annual Meeting abstract.