PAX6D instructs neural retinal specification from human embryonic stem cell-derived neuroectoderm.

Area of Science:

• Developmental biology
• Stem cell biology
• Ophthalmology

Background:

• Paired-box protein 6 (PAX6) is vital for neural retina and forebrain development.
• The specific role of PAX6 isoforms in neural retina specification is not fully understood.

Purpose of the Study:

• To investigate the function of PAX6 isoform D (PAX6D) in neural retina specification.
• To elucidate the molecular mechanism by which PAX6D directs retinal cell fate.

Main Methods:

• Utilized human embryonic stem cells (hESCs) for retinal cell differentiation.
• Employed CRISPR/Cas9 for PAX6D knockout and rescue experiments.
• Performed Chromatin Immunoprecipitation sequencing (ChIP-Seq) to identify PAX6D target genes.
• Conducted functional assays including WNT pathway inhibition and activation.

Main Results:

• PAX6D is expressed in neural retina cells during human eye development and hESC differentiation.
• hESCs lacking PAX6D failed to specify into neural retina cells.
• Induced expression of PAX6D, but not PAX6A, restored neural retina specification capacity.
• PAX6D directly targets retinal and non-retinal neural genes, including WNT8B.
• WNT8B acts as a critical mediator; its inhibition rescues PAX6D-deficient neuroepithelia, while its activation blocks retinal specification.

Conclusions:

• PAX6D is essential and sufficient for specifying neuroepithelia to neural retina cells.
• PAX6D exerts its function by regulating the WNT8B signaling pathway, thereby controlling retinal cell fate determination.