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Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium
Elizabeth E Capowski1, Joseph M Simonett1, Eric M Clark1
1Waisman Center.
Abstract:
Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.
Insights
Microphthalmia-associated transcription factor (MITF) is crucial for early human eye development. Its regulation by VSX2 impacts optic vesicle cell proliferation and retinal pigment epithelium maturation.
Area of Science:
- Developmental Biology
- Stem Cell Biology
- Ophthalmology
Background:
- Microphthalmia-associated transcription factor (MITF) regulates pigmented cell survival and differentiation.
- MITF is essential for mouse eye development, but its role in human retinogenesis is unknown.
- Human embryonic stem cells (hESCs) offer a model to study early human retinal development.
Purpose of the Study:
- Investigate the earliest roles of MITF in human retinogenesis using an hESC model.
- Determine MITF isoform expression patterns during retinal lineage differentiation.
- Examine the regulatory relationship between VSX2 and MITF isoforms.
Main Methods:
- Differentiated hESCs to recapitulate optic vesicle specification, RPE, and NRPC formation.
- Analyzed MITF isoform expression during retinal development.
- Investigated VSX2 targeting of MITF promoters.
- Manipulated MITF RNA and protein levels to assess functional consequences.
Main Results:
- MITF isoforms showed expression patterns similar to mouse models during hESC differentiation.
- Visual Systems Homeobox 2 (VSX2) directly targeted promoters of MITF-A, -D, and -H isoforms.
- Altered MITF levels led to decreased eye field transcription factors, reduced optic vesicle cell proliferation, and disrupted RPE maturation.
Conclusions:
- MITF plays a critical role in early human retinogenesis, influencing optic vesicle development and RPE maturation.
- VSX2 directly regulates key MITF isoforms during human retinal development.
- This hESC model provides a platform for studying complex transcription factors in human eye development.
