Byron H. Hartman

Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear

During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea.

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.

Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution.

The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification.

Intrinsic regenerative potential of murine cochlear supporting cells

The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different non-sensory cell populations by fluorescence-activated cell sorting (FACS). All four populations displayed proliferative potential, but only lesser epithelial ridge and supporting cells robustly gave rise to hair cell marker-positive cells. These results suggest that cochlear supporting cells and cells of the lesser epithelial ridge show robust potential to de-differentiate into prosensory cells that proliferate and undergo differentiation in similar fashion to native prosensory cells of the developing inner ear.

Notch Activity Is Downregulated Just prior to Retinal Ganglion Cell Differentiation

The Notch signaling pathway is important at several stages of retinal development including the differentiation of retinal ganglion cells and Müller glia. The downstream effectors of Notch signaling, Hes1 and Hes5, have been shown to be critical in the retina as well. While Notch activity directly regulates Hes1 and Hes5 elsewhere in the nervous system, it has been unclear whether Hes1 and/or Hes5 are directly regulated by Notch activity in the developing retina. Here, we report that both Hes1 and Hes5 are directly regulated by Notch activity during retinal development. Using fluorescence-based Hes1 and Hes5 reporter constructs, we can monitor Notch activity in progenitor cells in the intact retina, and we find that Notch activity is downregulated just prior to retinal ganglion cell differentiation.

Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate.

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.

NeuroD1 Regulates Expression of Thyroid Hormone Receptor β2 and Cone Opsins in the Developing Mouse Retina

The correct patterning of opsin expression in cone photoreceptors is critical for normal color vision. Thyroid hormone, and one of its receptors [thyroid hormone receptor β2 (TRβ2)], is an important regulator of opsin expression during cone photoreceptor development. Mice have two genes, encoding medium-wavelength (M) and short-wavelength (S) cone opsins. Targeted deletion of TRβ2 leads to a uniform expression of S-opsin in all cone photoreceptors and a loss of M-opsin. The control of expression of TRβ2 is therefore central to cone differentiation, yet there is little known about its regulation in the retina. We now report that the proneural bHLH (basic helix-loop-helix) transcription factor, NeuroD1, is necessary for sustained expression of TRβ2 in immature cone photoreceptors. Mice deficient in NeuroD1 develop an opsin phenotype virtually identical with that of TRβ2-deficient mice: all cones express S-opsin, and none expresses M-opsin. The introduction of NeuroD1 into embryonic retinal explants from NeuroD1−/− mice restores TRβ2 expression. NeuroD1 binds an E-box in the intron control region of the TRβ2 gene that mediates cone-specific expression, suggesting that NeuroD1 is a critical contributory factor to the expression of TRβ2 in cones. These results thus connect the proneural pathway with opsin selection to ensure correct cone patterning during retinal development.

Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development

Delta gene expression in Drosophila is regulated by proneural basic helix–loop–helix (bHLH) transcription factors, such as acheate‐scute. In vertebrates, multiple Delta‐like and proneural bHLH genes are expressed during neurogenesis, especially in the retina. We recently uncovered a relationship between Acheate‐scute like 1 (Ascl1), Delta‐like genes, and Notch in chick retinal progenitors. Here, we report that mammalian retinal progenitors are also the primary source of Delta‐like genes, likely signaling through Notch among themselves, while differentiating neurons expressed Jagged2. Ascl1 is coexpressed in Delta‐like and Notch active progenitors, and required for normal Delta‐like gene expression and Notch signaling. We also reveal a role for Ascl1 in the regulation of Hes6, a proneurogenic factor that inhibits Notch signaling to promote neural rather than glial differentiation. Thus, these results suggest a molecular mechanism whereby attenuated Notch levels coupled with reduced proneurogenic activity in progenitors leads to increased gliogenesis and decreased neurogenesis in the Ascl1‐deficient retina. Developmental Dynamics 238:2163–2178, 2009.

Dll3 is expressed in developing hair cells in the mammalian cochlea

Notch mediates the process of lateral inhibition that controls the production of hair cells in the inner ear. Hair cells are known to express Notch ligands Dll1 and Jag2, which signal through Notch1 in adjacent supporting cells. However, recent genetic and pharmacological studies indicate that the level of Notch‐mediated lateral inhibition is greater than can be accounted for by Dll1 and Jag2. Here, we report that another Notch ligand, Dll3, is expressed in developing hair cells, in a pattern that overlaps that of Dll1 and Jag2. We analyzed the cochleae of Dll3pu mutant mice, but did not detect any abnormalities. However, earlier studies have demonstrated that there is functional redundancy among Notch ligands in cochlear development and loss of one ligand can be at least partially compensated for by another. Thus Dll3 may play a role in lateral inhibition similar to that of Dll1 and Jag2. Developmental Dynamics 236:2875–2883, 2007.

Identification and characterization of mouse otic sensory lineage genes

Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells.