Abstract
Purpose: :
To elucidate the interactions of MITF and Chx10 (also known as Vsx2) in an hESC model of human optic vesicle development.
Methods: :
The H1 and H9 hESC lines were differentiated to neuroretina as described (1). After 16 days of differentiation, hESC-derived neurospheres were floated and optic vesicle (OV) bodies were identified and manually purified. These were either 1) formaldehyde fixed to crosslink Chx10 to chromatin for ChIP analysis or 2) lysed for RNA isolation and RT-PCR to study isoform-specific MITF expression or 3) infected with lentiviral constructs delivering shRNA for either Chx10 (90% knock down (2)) or a scrambled control.
Results: :
MITF-A and MITF-H were the two predominant MITF isoforms expressed in human ES cell-derived OV cells. Cells expressing these isoforms adopted either a neuroretinal or an RPE cell fate depending on the subsequent expression of Chx10 (1). We used shRNA to reduce Chx10 mRNA levels in OV bodies, which caused a reciprocal rise in mRNA levels of MITF as well as downstream targets of MITF such as dopachrome tautomerase. ChIP analysis showed that Chx10 directly bound the H promoter in regions containing putative consensus binding sites but not in upstream regions. Putative MITF-A consensus sites did not appear to be occupied in hESC derived OV bodies nor did Chx10 bind to MITF-B promoter regions. MITF-B was expressed in hESC-derived RPE but appeared to be restricted to committed RPE cells as it was not expressed in hESC-derived OV cells.
Conclusions: :
Aspects of early human retinal development can be modeled by ESC differentiation protocols, providing access to molecular events that cannot otherwise be studied in vivo. We present evidence that the neuroretina vs. RPE fate decision in hESC-derived OV bodies are similar, but not identical, to rodent models.1. Meyer et al. (2009) PNAS 106: 166982. Livne-bar et al. (2006) PNAS 103: 4988
Keywords: retinal development • transcription factors • retinal pigment epithelium