Harald Luksch

Significance: Genetically modified animals are crucial for functional studies and translational biomedical research. However, the generation of genome-edited large animals is time consuming and inefficient. Here, we report the generation of transgenic pigs and chickens that ubiquitously express Cas9 nuclease. The functionality of Cas9 was demonstrated for different target genes, a variety of cell types, and in vivo for the heart and the developing brain. Genome editing can now easily be carried out in vivo in specific organs or tissues without the need to generate germline-modified animals. These Cas9 transgenic animals provide an innovative and efficient resource for in vivo genome editing in biomedical and agricultural sciences.

Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons. Significance statement: Genome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

Sharp wave ripples (SWR) represent one of the most synchronous population patterns in the mammalian brain. Although SWRs are highly conserved throughout mammalian evolution, the existence of SWRs in non-mammalian species remains controversial. We reexamined the existence of avian SWRs by recording the brain activity during sleep and under anesthesia in two species of birds, the zebra finch and the chicken. Electrophysiological recordings using silicon probes implanted in the avian telencephalon revealed highly dynamic switching between high and low delta phases during sleep. High delta phases were composed of large-amplitude, negative deflections (sharp waves) that coincided with a high frequency oscillation (ripple). Correlation analysis revealed that these events were highly synchronous and spanned a large anatomical range of the avian telencephalon. Finally, detailed spike analysis revealed that an increase in the population spiking activity coincided with the occurrence of SWRs, that this spiking activity occurred in specific sequences of spike patterns locked to the SWRs, and that the mean population spiking activity peaked prior to the trough of the negative deflection. These results provide the first evidence of avian SWRs during natural sleep and under anesthesia, and suggest that the evolutionary origin of SWR activity may precede the mammalian-sauropsid bifurcation.

Visual information in birds is to great extent processed in the optic tectum (TeO), a prominent laminated midbrain structure. Retinal input enters the TeO in its superficial layers, while output is limited to intermediate and deeper layers. In addition to visual information, the TeO receives multimodal input from the auditory and somatosensory pathway. The TeO gives rise to a major ascending tectofugal projection where neurons of tectal layer 13 project to the thalamic nucleus rotundus, which then projects to the entopallium. A second tectofugal projection system, called the accessory pathway, has however not been studied as thoroughly. Again, cells of tectal layer 13 form an ascending projection that targets a nucleus known as either the caudal part of the nucleus dorsolateralis posterior of the thalamus (DLPc) or nucleus uveaformis (Uva). This nucleus is known for multimodal integration and receives additional input from the lateral pontine nucleus (PL), which in turn receives projections from layer 8–15 of the TeO. Here, we studied a particular cell type afferent to the PL that consists of radially oriented neurons in layer 9. We characterized these neurons with respect to their anatomy, their retinal input, and the modulation of retinal input by local circuits. We found that comparable to other radial neurons in the tectum, cells of layer 9 have columnar dendritic fields and reach up to layer 2. Sholl analysis demonstrated that dendritic arborization concentrates on retinorecipient layers 2 and 4, with additional arborization in layers 9 and 10. All neurons recorded in layer 9 received retinal input via glutamatergic synapses. We analyzed the influence of modulatory circuits of the TeO by application of antagonists to γ-aminobutyric acid (GABA) and acetylcholine (ACh). Our data show that the neurons of layer 9 are integrated in a network under strong GABAergic inhibition, which is controlled by local cholinergic activation. Output to the PL and to the accessory tectofugal pathway thus appears to be under strict control of local tectal networks, the relevance of which for multimodal integration is discussed.

Background: The epithelial layer of the nasal mucosa is the first barrier for drug permeation during intranasal drug delivery. With increasing interest for intranasal pathways, adequate in vitro models are required. Here, porcine olfactory (OEPC) and respiratory (REPC) primary cells were characterised against the nasal tumour cell line RPMI 2650. Methods: Culture conditions for primary cells from porcine nasal mucosa were optimized and the cells characterised via light microscope, RT-PCR and immunofluorescence. Epithelial barrier function was analysed via transepithelial electrical resistance (TEER), and FITC-dextran was used as model substance for transepithelial permeation. Beating cilia necessary for mucociliary clearance were studied by immunoreactivity against acetylated tubulin. Results: OEPC and REPC barrier models differ in TEER, transepithelial permeation and MUC5AC levels. In contrast, RPMI 2650 displayed lower levels of MUC5AC, cilia markers and TEER, and higher FITC-dextran flux rates. Conclusion: To screen pharmaceutical formulations for intranasal delivery in vitro, translational mucosal models are needed. Here, a novel and comprehensive characterisation of OEPC and REPC against RPMI 2650 is presented. The established primary models display an appropriate model for nasal mucosa with secreted MUC5AC, beating cilia and a functional epithelial barrier, which is suitable for long-term evaluation of sustained release dosage forms.

Editorial: The Superior Colliculus/Tectum: Cell Types, Circuits, Computations, Behaviors

Lugaro cells are inhibitory interneurons found in the upper granular layer of the cerebellar cortex, just below or within the Purkinje cell layer. They are characterized by (1) a fusiform soma oriented in the parasagittal plane, (2) two pairs of dendrites emanating from opposite ends of the soma, (3) innervation from Purkinje cell collaterals, and (4) an axon that projects into the molecular layer akin to granular cell parallel fibers. Lugaro cells have been described in mammals, but not in other vertebrate classes, save one report in teleost fish. Here, we propose the existence of Lugaro cells in the avian cerebellum based on the morphological characteristics and connectivity described above. Immunohistochemical staining against the calcium binding protein secretagogin (SCGN) revealed Lugaro-like cells in the pigeon cerebellum. Some SCGN-labeled cells exhibit fusiform somata and dendrites parallel to the Purkinje cell layer in the parasagittal plane, as well as long axons that project into the molecular layer and travel alongside parallel fibers in the coronal plane. While mammalian Lugaro cells are known to express calretinin, the SCGN-labeled cells in the pigeon do not. SCGN-labeled cells also express glutamic acid decarboxylase, confirming their inhibitory function. Calbindin labeling revealed Purkinje cell terminals surrounding the SCGN-expressing cells. Our results suggest that Lugaro cells are more widespread among vertebrates than previously thought and may be a characteristic of the cerebellum of all vertebrates.

Echolocating bats are known to fly and forage in complete darkness using the echoes of their actively emitted calls to navigate and to detect prey. However, under dim light conditions many bats can also rely on vision. Many flying animals have been shown to navigate by optic flow information, and recently, bats were shown to exploit echo-acoustic flow to navigate through dark habitats. Here we show for the bat Phyllostomus discolor that in lighted habitats where self-motion induced optic flow is strong, optic and echo-acoustic flow interact in their efficiency to guide navigation. Echo-acoustic flow showed a surprisingly strong effect compared to optic flow. We thus demonstrate multimodal interaction between two far-ranging spatial senses, vision and echolocation, available in this combination almost exclusively for bats and toothed whales. Our results highlight the importance of merging information from different sensory systems in a sensory-specialist animal to successfully navigate and hunt under difficult conditions.

Background: The use of therapeutic antibodies for the treatment of neurological diseases is of increasing interest. Nose-to-brain drug delivery is one strategy to bypass the blood brain barrier. The neonatal Fc receptor (FcRn) plays an important role in transepithelial transcytosis of immunoglobulin G (IgG). Recently, the presence of the FcRn was observed in nasal respiratory mucosa. The aim of the present study was to determine the presence of functional FcRn in olfactory mucosa and to evaluate its role in drug delivery. Methods: Immunoreactivity and messenger RNA (mRNA) expression of FcRn was determined in ex vivo porcine olfactory mucosa. Uptake of IgG was performed in a side-by-side cell and analysed by immunofluorescence. Results: FcRn was found in epithelial and basal cells of the olfactory epithelium as well as in glands, cavernous bodies and blood vessels. Allogenic porcine IgGs were found time-dependently in the lamina propria and along axonal bundles, while only small amounts of xenogenic human IgGs were detected. Interestingly, lymphoid follicles were spared from allogenic IgGs. Conclusion: Fc-mediated transport of IgG across the nasal epithelial barrier may have significant potential for intranasal delivery, but the relevance of immune interaction in lymphoid follicles must be clarified to avoid immunogenicity.