Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1×10 11 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1×10 12 vg AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust co-transduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell type-specific promoters, these AAVs provide targeted gene expression across the nervous system and enable efficient and versatile gene manipulation throughout the nervous system of transgenic and non-transgenic animals.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Mechanosensory hair cells (HCs) are the primary receptors of our senses of hearing and balance. Elucidation of the transcriptional networks regulating HC fate determination and differentiation is crucial not only to understand inner ear development but also to improve cell replacement therapies for hearing disorders. Here, we show that combined expression of the transcription factors Gfi1, Pou4f3 and Atoh1 can induce direct programming towards HC fate, both during in vitro mouse embryonic stem cell differentiation and following ectopic expression in chick embryonic otic epithelium. Induced HCs (iHCs) express numerous HC-specific markers and exhibit polarized membrane protrusions reminiscent of stereociliary bundles. Transcriptome profiling confirms the progressive establishment of a HC-specific gene signature during in vitro iHC programming. Overall, this work provides a novel approach to achieve robust and highly efficient HC production in vitro, which could be used as a model to study HC development and to drive inner ear HC regeneration.

Zombie active sites are only found in the cells were they are made, whereas the individual transcripts can diffuse away and be detected in cells other than their cell of origin. (a) Co-culture of mES-Z1 cells with non-transgenic parental cells. Active sites are only found in CFP+ cells. However, small diffraction limited dots are found in all cells including the non-transgenic ones. (b) Same image as a, but with blue and cyan channels turned off for better visibility of the barcode signal. A few examples of individual barcode transcripts are marked by circles. The inset shows a magnified and contrast adjusted view of a CFP negative cell, marked by the square, which contains some small barcode dots, indicating diffusion of individual RNA molecules from the cells in which they are produced. (c) In the absence of any transgenic cells, background non-specific signal is low. Indicating that the signal observed in the presence of transgenic cells is not non-specific HCR amplification. (d) Same image as c, but with blue and cyan channels turned off to make the lack of small dots more evident. The experiments were independently repeated three times with similar results. Scale bar is 25 μm. a b c d zBC Cerulean (mRNA) Cerulean (protein) DAPI zBC Cerulean (mRNA)

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. It originates from the otic placode, which invaginates, forming the otic vesicle; the latter gives rise to neurosensory and nonsensory elements of the adult membranous labyrinth. A hypothesis based on descriptive and experimental evidence suggests that the acquisition of discrete sensory patches during evolution of this primordium may be related to subdivision of an early pansensory domain. In order to gain insight into this developmental mechanism, we carried out a detailed analysis of the spatial and temporal expression pattern of the gene Fgf10, by comparing different markers of otic patterning and hair cell differentiation. Fgf10 expression labels a sensory-competent domain included in a Serrate-positive territory from which most of the sensory epithelia arise. Our data show that Fgf10 transcripts are present initially in a narrow ventromedial band of the rudimentary otocyst, extending between its rostral and caudal poles. During development, this Fgf10-expressing area splits repetitively into several separate subareas, creating six of the eight sensory organs present in birds. Only the lateral crista and the macula neglecta were initially Fgf10 negative, although they activated Fgf10 expression after their specification as sensory elements. These results allowed us to determine a timetable of sensory specification in the developing chick inner ear. The comparison of the expression pattern of Fgf10 with those of other markers of sensory differentiation contributes to our understanding of the mechanism by which vertebrate inner ear prosensory domains have arisen during evolution.