Origin of Neuroblasts in the Avian Otic Placode and Their Distributions in the Acoustic and Vestibular Ganglia.

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. This intricate sensory organ originates from the otic placode, which generates the sensory elements of the membranous labyrinth, as well as all the ganglionic neuronal precursors. How auditory and vestibular neurons establish their fate identities remains to be determined. Their topological origin in the incipient otic placode could provide positional information before they migrate, to later segregate in specific portions of the acoustic and vestibular ganglia. To address this question, transplants of small portions of the avian otic placode were performed according to our previous fate map study, using the quail/chick chimeric graft model. All grafts taking small areas of the neurogenic placodal domain contributed neuroblasts to both acoustic and vestibular ganglia. A differential distribution of otic neurons in the anterior and posterior lobes of the vestibular ganglion, as well as in the proximal, intermediate, and distal portions of the acoustic ganglion, was found. Our results clearly show that, in birds, there does not seem to be a strict segregation of acoustic and vestibular neurons in the incipient otic placode.

CRABP-I Expression Patterns in the Developing Chick Inner Ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions, regarded as an excellent system for analyzing events that occur during development, such as patterning, morphogenesis, and cell specification. Retinoic acid (RA) is involved in all these development processes. Cellular retinoic acid-binding proteins (CRABPs) bind RA with high affinity, buffering cellular free RA concentrations and consequently regulating the activation of precise specification programs mediated by particular regulatory genes. In the otic vesicle, strong CRABP-I expression was detected in the otic wall's dorsomedial aspect, where the endolymphatic apparatus develops, whereas this expression was lower in the ventrolateral aspect, where part of the auditory system forms. Thus, CRABP-I proteins may play a role in the specification of the dorsal-to-ventral and lateral-to-medial axe of the otic anlagen. Regarding the developing sensory patches, a process partly involving the subdivision of a ventromedial pro-sensory domain, the CRABP-I gene displayed different levels of expression in the presumptive territory of each sensory patch, which was maintained throughout development. CRABP-I was also relevant in the acoustic-vestibular ganglion and in the periotic mesenchyme. Therefore, CRABP-I could protect RA-sensitive cells in accordance with its dissimilar concentration in specific areas of the developing chick inner ear.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cyp1B1 expression patterns in the developing chick inner ear.

BACKGROUND: Retinoic acid (RA) plays an important role in organogenesis as a paracrine signal through transcriptional regulation of an increasing number of known downstream target genes, regulating cell proliferation, and differentiation. During the development of the inner ear, RA directly governs the morphogenesis and specification processes mainly by means of RA-synthesizing retinaldehyde dehydrogenase (RALDH) enzymes. Interestingly, CYP1B1, a cytochrome P450 enzyme, is able to mediate the oxidative metabolisms also leading to RA generation, its expression patterns being associated with many known sites of RA activity. RESULTS: This study describes for the first time the presence of CYP1B1 in the developing chick inner ear as a RALDH-independent RA-signaling mechanism. In our in situ hybridization analysis, Cyp1B1 expression was first observed in a domain located in the ventromedial wall of the otic anlagen, being included within the rostralmost aspect of an Fgf10-positive pan-sensory domain. As development proceeds, all identified Fgf10-positive areas were Cyp1B1 stained, with all sensory patches being Cyp1B1 positive at stage HH34, except the macula neglecta. CONCLUSIONS: Cyp1B1 expression suggested a possible contribution of CYP1B1 action in the specification of the lateral-to-medial and dorsal-to-ventral axes of the developing chick inner ear.

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Origin of acoustic-vestibular ganglionic neuroblasts in chick embryos and their sensory connections.

The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, which generates the sensory elements of the membranous labyrinth and all the ganglionic neuronal precursors. Neuroblast specification is the first cell differentiation event. In the chick, it takes place over a long embryonic period from the early otic cup stage to at least stage HH25. The differentiating ganglionic neurons attain a precise innervation pattern with sensory patches, a process presumably governed by a network of dendritic guidance cues which vary with the local micro-environment. To study the otic neurogenesis and topographically-ordered innervation pattern in birds, a quail-chick chimaeric graft technique was used in accordance with a previously determined fate-map of the otic placode. Each type of graft containing the presumptive domain of topologically-arranged placodal sensory areas was shown to generate neuroblasts. The differentiated grafted neuroblasts established dendritic contacts with a variety of sensory patches. These results strongly suggest that, rather than reverse-pathfinding, the relevant role in otic dendritic process guidance is played by long-range diffusing molecules.

Lineage does not regulate the sensory synaptic input of projection neurons in the mouse olfactory bulb.

Lineage regulates the synaptic connections between neurons in some regions of the invertebrate nervous system. In mammals, recent experiments suggest that cell lineage determines the connectivity of pyramidal neurons in the neocortex, but the functional relevance of this phenomenon and whether it occurs in other neuronal types remains controversial. We investigated whether lineage plays a role in the connectivity of mitral and tufted cells, the projection neurons in the mouse olfactory bulb. We used transgenic mice to sparsely label neuronal progenitors and observed that clonally related neurons receive synaptic input from olfactory sensory neurons expressing different olfactory receptors. These results indicate that lineage does not determine the connectivity between olfactory sensory neurons and olfactory bulb projection neurons.

Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.

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 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction 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 and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.

Fgf3 and Fgf16 expression patterns define spatial and temporal domains in the developing chick inner ear.

The inner ear is a morphologically complex sensory structure with auditory and vestibular functions. The developing otic epithelium gives rise to neurosensory and non-sensory elements of the adult membranous labyrinth. Extrinsic and intrinsic signals manage the patterning and cell specification of the developing otic epithelium by establishing lineage-restricted compartments defined in turn by differential expression of regulatory genes. FGF3 and FGF16 are excellent candidates to govern these developmental events. Using the chick inner ear, we show that Fgf3 expression is present in the borders of all developing cristae. Strong Fgf16 expression was detected in a portion of the developing vertical and horizontal pouches, whereas the cristae show weaker or undetected Fgf16 expression at different developmental stages. Concerning the rest of the vestibular sensory elements, both the utricular and saccular maculae were Fgf3 positive. Interestingly, strong Fgf16 expression delimited these Fgf16-negative sensory patches. The Fgf3-negative macula neglecta and the Fgf3-positive macula lagena were included within weakly Fgf16-expressing areas. Therefore, different FGF-mediated mechanisms might regulate the specification of the anterior (utricular and saccular) and posterior (neglecta and lagena) maculae. In the developing cochlear duct, dynamic Fgf3 and Fgf16 expression suggests their cooperation in the early specification and later cell differentiation in the hearing system. The requirement of Fgf3 and Fgf16 genes in endolymphatic apparatus development and neurogenesis are discussed. Based on these observations, FGF3 and FGF16 seem to be key signaling pathways that control the inner ear plan by defining epithelial identities within the developing otic epithelium.

Determination of the connectivity of newborn neurons in mammalian olfactory circuits.

The mammalian olfactory bulb is a forebrain structure just one synapse downstream from the olfactory sensory neurons and performs the complex computations of sensory inputs. The formation of this sensory circuit is shaped through activity-dependent and cell-intrinsic mechanisms. Recent studies have revealed that cell-type specific connectivity and the organization of synapses in dendritic compartments are determined through cell-intrinsic programs already preset in progenitor cells. These progenitor programs give rise to subpopulations within a neuron type that have distinct synaptic organizations. The intrinsically determined formation of distinct synaptic organizations requires factors from contacting cells that match the cell-intrinsic programs. While certain genes control wiring within the newly generated neurons, other regulatory genes provide intercellular signals and are only expressed in neurons that will form contacts with the newly generated cells. Here, the olfactory system has provided a useful model circuit to reveal the factors regulating assembly of the highly structured connectivity in mammals.

Expression patterns of Irx genes in the developing chick inner ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. The molecular patterning of the developing otic epithelium creates various positional identities, consequently leading to the stereotyped specification of each neurosensory and non-sensory element of the membranous labyrinth. The Iroquois (Iro/Irx) genes, clustered in two groups (A: Irx1, Irx2, and Irx4; and B: Irx3, Irx5, and Irx6), encode for transcriptional factors involved directly in numerous patterning processes of embryonic tissues in many phyla. This work presents a detailed study of the expression patterns of these six Irx genes during chick inner ear development, paying particular attention to the axial specification of the otic anlagen. The Irx genes seem to play different roles at different embryonic periods. At the otic vesicle stage (HH18), all the genes of each cluster are expressed identically. Both clusters A and B seem involved in the specification of the lateral and posterior portions of the otic anlagen. Cluster B seems to regulate a larger area than cluster A, including the presumptive territory of the endolymphatic apparatus. Both clusters seem also to be involved in neurogenic events. At stages HH24/25-HH27, combinations of IrxA and IrxB genes participate in the specification of most sensory patches and some non-sensory components of the otic epithelium. At stage HH34, the six Irx genes show divergent patterns of expression, leading to the final specification of the membranous labyrinth, as well as to cell differentiation.

piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice.

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR-/- mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR-/- than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR-/- males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR-/- ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.

Generation of sensory hair cells by genetic programming with a combination of transcription factors.

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.

Fate map of the chicken otic placode.

The inner ear is an intricate three-dimensional sensory organ that arises from a flat, thickened portion of the ectoderm termed the otic placode. There is evidence that the ontogenetic steps involved in the progressive specification of the highly specialized inner ear of vertebrates involve the concerted actions of diverse patterning signals that originate from nearby tissues, providing positional identity and instructive context. The topology of the prospective inner ear portions at placode stages when such patterning begins has remained largely unknown. The chick-quail model was used to perform a comprehensive fate mapping study of the chick otic placode, shedding light on the precise topological position of each presumptive inner ear component relative to the dorsoventral and anteroposterior axes of the otic placode and, implicitly, to the possible sources of inducing signals. The findings reveal the existence of three dorsoventrally arranged anteroposterior domains from which the endolymphatic system, the maculae and basilar papilla, and the cristae develop. This study provides new bases for the interpretation of earlier and future descriptive and experimental studies that aim to understand the molecular genetic mechanisms involved in otic placode patterning.

Fgf10 expression patterns in the developing chick inner ear.

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.

Distinct and redundant expression and transcriptional diversity of MEIS gene paralogs during chicken development.

Members of the Meis family of TALE homeobox transcription factors are involved in many processes of vertebrate development and morphogenesis, showing extremely complex transcriptional and spatiotemporal expression patterns. In this work, we performed a comprehensive study of chicken Meis genes using multiple approaches. First, we assessed whether the chicken genome contains a Meis3 ortholog or harbors only two Meis genes; we gathered several lines of evidence pointing to a specific loss of the Meis3 ortholog in an early ancestor of birds. Next, we studied the transcriptional diversity generated from chicken Meis genes through alternative splicing during development. Finally, we performed a detailed analysis of chick Meis1/2 expression patterns during early embryogenesis and organogenesis. We show that the expression of both Meis genes begins at the gastrulation stage in the three embryonic layers, presenting highly dynamic patterns with overlapping as well as distinct expression domains throughout development.

Meis gene expression patterns in the developing chicken inner ear.

We are interested in stable gene network activities operating sequentially during inner ear specification. The implementation of this patterning process is a key event in the generation of functional subdivisions of the otic vesicle during early embryonic development. The vertebrate inner ear is a complex sensory structure that is a good model system for characterization of developmental mechanisms controlling patterning and specification. Meis genes, belonging to the TALE family, encode homodomain-containing transcription factors remarkably conserved during evolution, which play a role in normal and neoplastic development. To gain understanding of the possible role of homeobox Meis genes in the developing chick inner ear, we comprehensively analyzed their spatiotemporal expression patterns from early otic specification stages onwards. In the invaginating otic placode, Meis1/2 transcripts were observed in the borders of the otic cup, being absent in the portion of otic epithelium closest to the hindbrain. As development proceeds, Meis1 and Meis2 expressions became restricted to the dorsomedial otic epithelium. Both genes were strongly expressed in the entire presumptive domain of the semicircular canals, and more weakly in all associated cristae. The endolymphatic apparatus was labeled in part by Meis1/2. Meis1 was also expressed in the lateral wall of the growing cochlear duct, while Meis2 expression was detected in a few cells of the developing acoustic-vestibular ganglion. Our results suggest a possible role of Meis assigning regional identity in the morphogenesis, patterning, and specification of the developing inner ear.

Raldh3 gene expression pattern in the developing chicken inner ear.

Retinoic acid (RA), an active metabolite of vitamin A, is a diffusible molecule that regulates the expression of several families of genes, playing a key role in specification processes during chordate development. With the aim of defining its possible role in the developing chick inner ear, we obtained in this work a detailed spatiotemporal distribution of the enzymes involved in its synthesis, the retinaldehyde dehydrogenases (RALH1-4). Our results showed that, in contrast to the mouse inner ear, Raldh3 expression was the only Raldh gene detected in the developing chick inner ear, where it appears as early as stage 18. During inner ear morphogenesis, Raldh3 expression was predominantly observed in the endolymphatic system. The Raldh3 expression pattern delimited totally or partially the Bmp4-positive presumptive territories of vestibular sensory epithelia by stage 24 and the basilar papilla at stage 34, suggesting a possible involvement of RA in their specification. In addition, several vestibular sensory areas showed some Raldh3-expressing cells close to the Raldh3-positive domain. These results suggest that the RA signaling pathway may play a role in the initial patterning of the otic epithelium and cell differentiation therein, providing local positional information. Having in mind this Raldh3 expression pattern, we discuss the regulatory interactions among the RA, bone morphogenetic protein, and fibroblast growth factor signaling pathways in the specification of otic sensory elements. Our investigation may underpin further experimental studies aimed at understanding the possible role of signaling pathways in patterning of the developing chick inner ear.