Hair-cell-specific Genes in the Embryonic Chicken Inner Ear by Overexpression

The inner ear houses organs used for hearing and balance that use hair cells to accomplish their tasks. Inner ear development remains to be fully understood, and advancing knowledge in development could lead to therapies and treatments for hearing problems. A gene called Atoh1 is necessary for hair cell formation and has been shown to increase hair cell number when overexpressed. Importantly, microRNAs from the 183 family (miRs-183, -182, and -96) are also expressed in developing hair cells. MicroRNAs are small RNA molecules that bind to messenger RNAs and prevent translation. MicroRNAs have been associated with cellular functions such as programmed cell death, cell division, and differentiation. In 2009, miR-96 was implicated in a congenital form of human deafness. Like Atoh1, miR-96 increases hair cell number when over-expressed. However, we do not know if the effects of overexpressing these genes will be additive. To address this, we plan to inject the inner ears of chicken embryos with a virus that causes overexpression of miRNAs from the 183 family and Atoh1. In order to establish the effects of injecting this virus, we are gathering data on control embryos injected with a non-bioactive virus to acquire a baseline for hair cell quantification. Hair cells will be quantified using image-analysis software after injection of the virus. The data collection methods are still being refined and we do not yet have any data. Further research potentially could utilize these results to develop therapeutic treatments for deafness

Zika Virus Can Strongly Infect and Disrupt Secondary Organizers in the Ventricular Zone of the Embryonic Chicken Brain

Zika virus (ZIKV) is associated with severe neurodeve- lopmental impairments in human fetuses, including microencephaly. Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in nonuniform periventricular infec- tion 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influ- ence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8 expressed at the midbrain basal plate, hypotha- lamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced, and a SHH-dependent cell popula- tion in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non- cell-autonomous changes in brain patterning

Expression and misexpression of the MIR-183 family in the developing hearing organ of the chicken

The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in nonsensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes. Copyright: © 2015 Zhang et al

Vertebrate Lrig3-ErbB Interactions Occur In Vitro but Are Unlikely to Play a Role in Lrig3-Dependent Inner Ear Morphogenesis

Background: The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes. Methodology/Principal Findings: Here, we report that Lrig3, like Lrig1, is able to interact with ErbB receptors in vitro. We examined the in vivo significance of these interactions in the inner ear, where Lrig3 controls semicircular canal formation by determining the timing and extent of Netrin1 expression in the otic vesicle epithelium. We find that ErbB2 and ErbB3 are present in the early otic epithelium, and that Lrig3 acts cell-autonomously here, as would be predicted if Lrig3 regulates ErbB2/B3 activity. However, inhibition of ErbB activation in the chick otic vesicle has no detectable effect on Netrin gene expression or canal morphogenesis. Conclusions/Significance: Our results suggest that although both Lrig1 and Lrig3 can interact with ErbB receptors in vitro, modulation of Neuregulin signaling is unlikely to contribute to Lrig3-dependent processes of inner ear morphogenesis. These results highlight the similar binding properties of Lrig1 and Lrig3 and underscore the need to determine how these two family members bind to and regulate different receptors to affect diverse aspects of cell behavior in vivo

Vertebrate Lrig3-ErbB Interactions Occur In Vitro but Are Unlikely to Play a Role in Lrig3-Dependent Inner Ear Morphogenesis.

The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo

Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans

Postcoloniality without race? Racial exceptionalism and south-east European cultural studies

The black Dutch feminist Gloria Wekker, assembling past and present everyday expressions of racialized imagination which collectively undermine hegemonic beliefs that white Dutch society has no historic responsibility for racism, writes in her book White Innocence that ‘one can do postcolonial studies very well without ever critically addressing race’ (p. 175). Two and a half decades after the adaptation of postcolonial thought to explain aspects of cultural politics during the break-up of Yugoslavia created important tools for understanding the construction of national, regional and socio-economic identities around hierarchical notions of ‘Europe’ and ‘the Balkans’ in the Yugoslav region and beyond, Wekker’s observation is still largely true for south-east European studies, where no intervention establishing race and whiteness as categories of analysis has reframed the field like work by Maria Todorova on ‘balkanism’ or Milica Bakić-Hayden on ‘symbolic geographies’ and ‘nesting orientalism’ did in the early 1990s. Critical race theorists such as Charles Mills nevertheless argue that ‘race’ as a structure of thought and feeling that legitimised colonialism and slavery (and still informs structural white supremacy) involved precisely the kind of essentialised link between people and territory that south-east European cultural theory also critiques: the construction of spatialised hierarchies specifying which peoples and territories could have more or less access to civilisation and modernity. South-east European studies’ latent racial exceptionalism has some roots in the race-blind anti-colonial solidarities of state socialist internationalism (further intensified for Yugoslavia through the politics of Non-Alignment) but also, this paper suggests, in deeper associations between Europeanness, whiteness and modernity that remain part of the history of ‘Europe’ as an idea even if, by the end of the 20th century, they were silenced more often than voiced