Tillotson Cell Biology Unit
Directed by Albert Edge, PhD, the Tillotson Cell Biology Unit is home to a team of scientists interested in regeneration in sensory systems and the complex molecular signaling in cell fate determination.
In the nervous system, the replacement of cells is challenging because of the complexity of the numerous cell types and the connections between them, and because there is little spontaneous repair after cell loss. The hair cells of the cochlea are epithelial cells, specialized for the detection of sound and for the transduction of sound to an electrical signal. These cells and their synapse, with afferent neurons, form a neural circuit whose development is choreographed to achieve the extraordinary sense of hearing over a broad dynamic range at a defined set of frequencies. Our work on the molecular cues in this system has begun to define the steps needed for differentiation of hair cells and neurons of the auditory system, and the regenerative biology of the system is poised for significant progress. We have identified cells that act as progenitors for both sensory hair cells and neurons and that may allow rebuilding of damaged sensory circuits. We have also made progress in studying mechanisms of synaptogenesis.
To accomplish this, we study cell fate determination both during development and regeneration. We are exploring the role of epigenetic silencing of proneural genes in the adult. We use CRISPR and siRNA screening to investigate the expression of genes during differentiation of stem cells to hair cells and neurons to understand which genes are necessary to obtain fully differentiated cells.
Areas of Research
Hair cell regeneration
The sensitive cells of the inner ear are not replaced after damage in mammals, unlike the sensory cells of lower vertebrates that regenerate spontaneously when lost to injury. We have recently shown, however, that newborn mammals retain the regenerative capacity seen in lower vertebrates. Regeneration is driven by Wnt signaling, based on gene expression profiles, and is inhibited by blocking the Wnt signal. The regenerative response is lost as the animal ages, but Wnt signaling is maintained.
We showed that a subset of supporting cells in the cochlea, identified by their expression of Lgr5, a gene first identified as a marker for stem cells in the epithelium of the intestine act as stem cells. The cochlear stem cells that are the source of regenerating sensory cells in the newborn sensory epithelium and account for the hair cell regenerating capacity of the cochlea are Lgr5 supporting cells. These Lgr5-expressing progenitors can be recruited for regeneration and have the capacity to generate organoids from single cochlear cells. The identification of Lgr5-expressing cells and their expansion in organoids provides a tool for assessing gene expression during stem cell expansion and differentiation to hair cells. These cells are not active under normal conditions, but can be recruited by activation of Wnt signaling. These cells are stimulated to divide by β-catenin in the post-natal cochlea. Wnt signaling, in fact, converts Lgr5 cells to hair cells by stimulating transcription factor Atoh1.
Through our investigations, we have found that hair cells in the adult, as in the newborn animal, can be generated from surrounding supporting cells by manipulation of signaling pathways. We have shown that hair cells can be generated from supporting cells by inhibition of Notch in the adult cochlea after damage, leading to a partial recovery of hearing in deafened mice. The drugs used to inhibit the Notch pathway are an example of the use of inhibitors of critical signaling pathways to achieve reprogramming. This took advantage of the early discovery that Atoh1 upregulation could achieve reprogramming in an appropriate background. Atoh1 is one of the first reprogramming factors and its ability to convert supporting cells to hair cells clearly showed the power of these factors to determine cell fate.
What changes with increasing age are epigenetic marks that restrict the capacity of these cells to respond to Wnt signaling. Modifications of histones activate genes by altering the structure of nucleosomes and converting open segments of DNA into closed configurations, thus regulating transcription at the epigenetic level. The methylation of histones at specific gene enhancers is translated into changes in acetylation of histones, which increases the open configuration of DNA. Inhibitors of histone deacetylase (HDAC) increase acetylation of histones and lead directly to the opening of DNA. Epigenetics, combined with gene editing, enables access to the DNA of specific genes necessary for converting stem cells in the adult ear to hair cells. The discoveries on transcriptional regulation of cell fate have far-reaching importance for stem cell biology.
Because of the important role it plays in generation of hair cells, our aim is to understand upstream signaling pathways leading to the expression of Atoh1 with the long-term goal of generating new hair cells and developing treatments for hearing loss. Epigenetic changes prevent the activation of proneural genes in response to Wnt and Notch signaling in the differentiation of hair cells. We are developing drugs that can drive the differentiation of new sensory cells by targeting the pathways and signaling molecules in cochlear stem cells. Loss of sensory cells in the inner ear due to excessive noise, drugs, disease, or aging results in deafness. This focus on regeneration of sensory cells is highly relevant to a significant translation goal, the replacement of sensory hair cells for the treatment of hearing loss.
Similar to regeneration of hair cells, regeneration of auditory neurons does not occur in adults. We have shown that peripheral axons of spiral ganglion neurons from newborn animals can extend their processes to hair cells, where they are able to form synapses after removal of the afferent innervation or damage to the afferent synapse. To learn more about how these synapses form, we have developed an in vitro assay for synaptogenesis. We are using this system to understand mechanisms guiding neuron growth and formation of synapses with hair cells. Questions about the rebuilding of neural circuits can be answered in this defined system. Genetic manipulation of gene expression is being used to test for key molecules for the auditory neurons to connect to the hair cells, recapitulating the original innervation.
We have been able to determine the key steps in differentiation of auditory neurons from embryonic stem cells and iPS cells. We have shown that afferent auditory neural fate is controlled by bHLH transcription factor, Ngn1, activated by upstream Sox2 signaling, and we have found that sensory neurons with characteristics of spiral ganglion cells can be induced by drug and growth factor treatment of human embryonic stem cells.
We have produced neural progenitors from mouse and human embryonic stem cells that, when grafted into the inner ear of animal models deafened by the ablation of neurons, rebuild neural circuits and form synapses with the sensory cells in the adult, where neural loss is irreversible. We have demonstrated partial recovery of hearing in animals deafened by loss of afferent neurons after transplantation of neurons made from embryonic stem cells and regrowth of neurons to hair cells.
The acquisition of neural cell fate is significant because loss of both neurons and hair cells in the cochlea are major causes of deafness. Insights into rebuilding of neural circuits will have application to neural regeneration after loss of cells to neurodegenerative processes. Regeneration of auditory neurons and their connections to hair cells could be a treatment for hearing loss.
About Albert Edge, PhD
Dr. Edge earned his doctorate in biochemistry and molecular biology at Albany Medical College and completed a postdoctoral fellowship in the Department of Biological Chemistry at Harvard Medical School, where he was an Iacocca Fellow and a Capps Scholar. Prior to joining Mass. Eye and Ear in 2003, Dr. Edge was an Assistant Professor at Harvard Medical School and the Joslin Diabetes Center. He is now a Professor at Harvard Medical School and the Director of the Tillotson Cell Biology Unit of the Eaton-Peabody Laboratories at Mass. Eye and Ear. He is a member of the Speech and Hearing Biosciences and Technology program faculty and has been appointed Distinguished Visiting Professor at Keio University School of Medicine. Learn more.