Abstract
The intrinsically photosensitive retinal ganglion cells (ipRGC) are the conduit between the retina and brain regions responsible for non-image-forming and image-forming vision. In mice, six ipRGC subtypes have been discovered based on morphological characteristics, functions, and molecular profiles. All ipRGCs arise from Tbr2-expressing RGCs during developmental stages and subsequently diverge and differentiate into the six mature, distinct subtypes in adult retinas. However, the cellular and molecular mechanisms controlling the formation and maturation of the six ipRGC subtypes remain elusive. Here, we demonstrate that two Tbr2-dependent transcription factors, Iroquois‑related homeobox 1 (Irx1) and T-box containing factor 20 (Tbx20), are key downstream transcription factors guiding lineage segregations of Tbr2-expressing RGC into distinct adult ipRGC subtypes. Both factors also control Opn4 expression. Irx1 is expressed in the M3, M4, and M5 subtypes, while Tbx20 is predominantly expressed in M1, M2, M6, and subgroups of M3 and M5. When Irx1 is ablated during retinal development, Opn4 expression is significantly reduced in the M3, M4, and M5 ipRGC groups; however, the formation of Irx1-expressing ipRGCs is not affected. In contrast, when Tbx20 is deleted, a significant number of Tbx20-expressing cells fail to develop while Opn4 expression is down-regulated. These findings reveal two parallel transcription cascades downstream of Tbr2 for controlling ipRGC subtype formation, fate divergence, and maintenance in the adult retina.
Data availability
The raw datasets and normalized count data for the bulk RNA-seq have been deposited in the NCBI Gene Expression Omnibus repository (GSE149388). The source data can be available in the supplementary data.
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Acknowledgements
This work was supported by grants from the National Institutes of Health to C.-A.M. (EY024376), C.-K.C (EY032898, EY034219), and B.A.A. (DE028527, DE026433, T90DE023520). This work was also supported in part by National Eye Institute Vision Core Grants P30EY028102 (UTHealth), the Hermann Eye Fund (UTHealth), and the Stephen Lasher Endowed Professorship (UTHealth). We acknowledge Dr. Jan Parker-Thornburg and the Genetically Engineered Mouse Facility at The University of Texas MD Anderson Cancer Center for making Irx1HA3-P2ACreERT2/+ and Tbx20HA3-P2ACreERT2/+ mouse lines. Irx1flox mouse was generated by GemPharmatech USA (San Diego, CA; Strain #: T008697). Tbx20flox was purchased from Jackson Lab (Bar Harbor, ME; Strain #:024665; RRID: IMSR_JAX:024665). Six3-Cre was a gift from Dr. Yas Furuta. Opn4Cre was a gift from Dr. Samer Hattar. RosaiAP was a gift from Dr. Tudor Badea. We thank Dr. Karthik Shekhar for sharing unpublished data and his valuable suggestions on scRNA-seq clustering. We thank Andrew Kim (UT McGovern Medical School) and Amber Lewis (UTHSC at San Antonio) for technical assistance.
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T.K., C.K.C., and C.A.M. conceptualized and designed experiments. T.K., C.K.C., H.Y.A., D.S., S.E., B.A.A., L.S., Y.J.C., and C.A.M. executed experiments. C.A.M. and C.K.C. wrote the paper.
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Kiyama, T., Chen, CK., Altay, H.Y. et al. Tbr2-dependent parallel pathways regulate the development of distinct ipRGC subtypes. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09645-4
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DOI: https://doi.org/10.1038/s42003-026-09645-4
