Cochlear Organoids Reveal HIC1’s Role in Hair Cell Differentiation
By Dunia Abdul-Aziz, M.D.
Sensory hair cells in the cochlea translate sound into electrical signals that they transmit to auditory neurons. These cells are lost over the course of life through cumulative damage from aging, infection, certain medications, and loud sounds. Sensory hair cells do not grow back and consequently hearing loss is irreversible.
In contrast, non-mammals, like birds and fish, can regenerate their damaged hair cells. Recent studies have also found spontaneous regeneration of hair cells after damage in the newborn mouse cochlea and balance organ. This regeneration involves a series of molecular pathways whereby precursor progenitor/stem cells (which are a subset of supporting cells that reside adjacent to the hair cells) begin to express the key hair cell transcription factor ATOH1 and differentiate into hair cells. However, this capacity for regeneration disappears during maturation of the mammalian cochlea, suggesting that regeneration is possible but repressed after development.
Understanding this molecular blockade hindering progenitor cell to hair cell conversion promises to reveal key steps needed to reverse hearing loss. Studying these pathways has traditionally been limited by complex transgenic mice and a few sets of relevant tools in a dish. The advent of inner ear organoids, which yields an expanded pool of progenitor cells, revolutionized our ability to study this process robustly in a dish. We derive organoids by first isolating and expanding progenitor cells from newborn mice cochleae and then differentiate them to hair cells. We can genetically modify the organoids to study how specific proteins affect this process.
Representative brightfield image of inner ear organoids. Within the organoids, a much larger proportion of cells in which Hic1 is knocked down (short-hairpin RNA to Hic1 (shHic1), marked by a red reporter) demonstrate overlapping expression of Atoh1 (green), as compared to untreated organoids or organoids that are treated with non-targeting shRNA. In our study, we further show that these cells express other hair cell markers consistent with their differentiation into hair cells.
We focused on the hypermethylated in cancer 1 (HIC1) protein as its role regulating Atoh1 expression has been established in other systems. It has been previously shown to directly bind to and suppress the regulatory regions around the Atoh1 gene during cerebellar development, and Hic1 deletion appears to permit Atoh1 expression and differentiation of Paneth cells in the intestine (like hair cells, Paneth cells express Atoh1). We hypothesized that HIC1 could contribute to repression of the Atoh1 gene in the cochlea through transcriptional regulation and interaction with Wnt, a key signaling pathway important in cochlear development.
As we reported in Stem Cell Reports in April 2021, we found that across various time points (ages), Hic1 is expressed throughout the mouse sensory epithelium. In cochlear organoids, HIC1 knockdown (suppression) induces Atoh1 expression and promotes hair cell differentiation (see the figure above), while HIC1 overexpression hinders differentiation.
We go on to study HIC1’s interaction with Wnt signaling which appears to be important to its mechanism. Our findings reveal the importance of HIC1 repression of Atoh1 in the cochlea. It also demonstrates the power of combining the organoid model with the genetic toolkit to study key regulators of hair cell differentiation, which we hope will be leveraged to advance our understanding of hair cell development and regeneration.
A 2019 Emerging Research Grants scientist, Dunia Abdul-Aziz, M.D., is an otolaryngologist at Mass Eye and Ear and an instructor in otolaryngology–head and neck surgery at Harvard Medical School. For more, see hhf.org/erg and hhf.org/mtr.