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

Recasting the theory of mosquito-borne pathogen transmission dynamics and control

Mosquito-borne diseases pose some of the greatest challenges in public health, especially in tropical and sub-tropical regions of theworld. Efforts to control these diseases have been underpinned by a theoretical framework developed for malaria by Ross and Macdonald, including models, metrics for measuring transmission, and theory of control that identifies key vulnerabilities in the transmission cycle. That framework, especially Macdonald\u27s formula for R0 and its entomological derivative, vectorial capacity, are nowused to study dynamics and design interventions for many mosquito-borne diseases. A systematic review of 388 models published between 1970 and 2010 found that the vast majority adopted the Ross-Macdonald assumption of homogeneous transmission in a well-mixed population. Studies comparing models and data question these assumptions and point to the capacity to model heterogeneous, focal transmission as the most important but relatively unexplored component in current theory. Fine-scale heterogeneity causes transmission dynamics to be nonlinear, and poses problems for modeling, epidemiology and measurement. Novel mathematical approaches show how heterogeneity arises from the biology and the landscape on which the processes of mosquito biting and pathogen transmission unfold. Emerging theory focuses attention on the ecological and social context formosquito blood feeding, themovement of both hosts and mosquitoes, and the relevant spatial scales for measuring transmission and for modeling dynamics and control

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days

Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences

MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use

Ectopic expression of miR-183 family is confirmed <i>in vivo</i> at S31 and S40.

<p>(A, B) miR-182 expression in left control and right pGFP-183F-transfected ears of a S31 embryo that was electroporated at S17. Immunostaining of GFP from adjacent sections through the right BP is shown in the right column. (A) Sections through the maculae show ectopic miR-182 signal in the right ear that overlaps with GFP immunolabeling adjacent to the saccular macula (arrows). Also, foci of stronger miR-182 signal in the vestibular ganglion correspond to regions showing GFP+ cells. (B) Sections through the BPs show GFP expression in (arrows) and adjacent to the sensory region that overlaps with foci of higher miR-182 signal. Ectopic expression is also present in the cochleolagenar ganglion. (C) miR-183 expression shown by in situ hybridization in left control and right pGFP-183F-electroporated BPs at S40 (electroporated at S11+), with GFP fluorescence for comparison in the right ear. Arrows point to examples where GFP+ cells superimpose with a higher intensity of signal for miR-183. Note the presence of GFP in the non-sensory epithelial cells (arrowheads) on both sides of the BP. These transfected cells are not visible in the image taken after in situ hybridization because these epithelial domains were removed for flat mounting. (D) Patches of overexpression in S40 BPs (electroporated at S11+, S12 and S14, respectively) are readily apparent for miR-183, miR-182 and miR-96. Separate images with focus on the HC layer versus SC layer are presented. Arrows point to ectopic expression in non-sensory epithelial cells. Scale bar in A and D equal 100μm. Scale bar in C equals 0.5mm.</p

miR-183 family is present in HCs in S38–S45 BPs.

<p>(A) The cartoon of THC and SHC distribution in the BP is modified from Tanaka and Smith, 1978 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132796#pone.0132796.ref005" target="_blank">5</a>]. (B-D) The expression of miR-182, miR-183 and miR-96 in the BPs from S38 to S45. The regions denoted by the arrowheads in B are shown at higher magnification in B’. (E) Cross section through S40- BP after in situ hybridization of miR-96 confirmed that miR-96 is only present in HCs, not in SCs. Scale bar in B-D equals 0.5mm. Scale bar in B’ and E equals 20μm.</p

Ectopic expression of miR-183 family can bias progenitor cells towards HC fate.

<p>(A) A S38 BP electroporated with pGFP-183F at S11- is immunostained with GFP and HCS-1. HCS-1 staining is present in the BP and the lagena, but not in GFP+ non-sensory epithelia (Hm: homogene cells; Hy: hyaline cells). (B) A S38 BP electroporated with pGFP-183F is stained with antibodies to GFP, HCS-1 and Sox2. Images through the layers of HCs and SCs are shown. Co-localization of Sox2 and HCS-1 is not observed. Arrows are pointing to a few GFP+ SC nuclei. Note that GFP+ SC nuclei have similar Sox2 levels as GFP-negative SC nuclei. Boxed areas are shown with higher magnification. (C) The number of GFP+ SCs and HCs are counted from BPs electroporated with pGFP-183F (n = 9) or pGFP (n = 8) and the ratio of GFP+ SCs/GFP+ HCs is calculated and compared (mean ± SEM). This ratio shows a significant difference (p < 0.05) by multiple t-tests between pGFP-183F and pGFP-electroporated samples only at the middle position along the longitudinal axis of the BP. 2-way ANOVA shows that the ratio is significantly affected by the positions along the BPs. Scale bar in A equals 100μm and in B equals 20μm.</p

Ectopic expression of miR-183 family does not alter HC fate along and across the BP.

<p>(A) A S40- BP electroporated with pGFP-183F at S11 is stained with GFP, phalloidin that labels actin and HC marker HCS-1. Images through the layer of HC bodies and stereocilia bundles at the base and the apex are shown. GFP+ basal HCs have similar cell cross-section areas and similar width of stereocilia bundles compared with their GFP-negative neighboring HCs, thus they do not gain the characteristics of apical HCs. (B) Quantification of maximum cross-section areas of HCs are done in the base of pGFP-183F transfected BPs. Tukey box and whiskers are shown here with outliers shown in dots. 2-way ANOVA showed no significant difference between GFP-negative HCs (GFP-) and GFP+ HCs. (C) A S40- BP electroporated with pGFP-183F at S15 is stained with GFP, CtBP2 that labels ribbon synapses and HCS-1. Maximum intensity projections of the image stack at the base of the BP are shown with neural on top. (D) Numbers of ribbon synapses in GFP+ SHCs, GFP-negative SHCs, GFP-negative THCs and GFP+ THCs from the base of pGFP-183F electroporated BPs (n = 5) are compared (mean ± SEM). The ribbon synapse numbers in GFP+ SHCs and GFP-negative SHCs are not significantly different by 2-way ANOVA analysis. Scale bar in A and C equals 20μm. Boxed areas are shown with high magnification. ND: not determined.</p

miR-182 is expressed in HCs and ganglion neurons of inner ear at S28 and S31.

<p>(A) Section in situ hybridization of miR-182 shows its presence in cristae and maculae at S28. (B) The expression of miR-182 in the HCs of vestibular organs at S31. C: The expression of miR-182 in the cochlear duct at S31. Sections across the base and the apex of the BP are shown. Note that HCs start to differentiate in the apex at this stage. A, anterior; AC, anterior crista; BP, basilar papilla; CG: cochleolagenar ganglion; M, medial; PC, posterior crista; SM, saccular macula; UM, utricular macula; VG, vestibular ganglion. Scale bar equals 100μm.</p