International audienceWe implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology. Cop. 2011 Nature America, Inc. All rights reserved

Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) gene project their axons to a few specific glomeruli that reside at recognizable locations in the olfactory bulb. Connecting approximately 1000 populations of OSNs to the approximately 1800 glomeruli of the mouse bulb poses a formidable wiring problem. Additional progress in understanding the mechanisms of neuronal connectivity is dependent on knowing how these axonal pathways are organized and how they form during development. Here we have applied a genetic approach to this problem. We have constructed by gene targeting novel strains of mice in which either all OSNs or those that express a specific OR gene, M72 or M71, also produce green fluorescent protein (GFP) or a fusion of tau with GFP. We visualized OSNs and their axons in whole mounts with two-photon laser scanning microscopy. The main conclusion we draw from the three-dimensional reconstructions is the high degree of morphological variability of mature glomeruli receiving axonal input from OR-expressing OSNs and of the pathways taken by the axons to those glomeruli. We also observe that axons of OR-expressing OSNs do not innervate nearby glomeruli in mature mice. Postnatally, a tangle of axons from M72-expressing OSNs occupies a large surface area of the bulb and coalesces abruptly into a protoglomerulus at a reproducible stage of development. These results differ in several aspects from those reported for the development of glomeruli receiving input from OSNs expressing the P2 OR, suggesting the need for a more systematic examination of OR-specific glomeruli.

The mammalian olfactory sense employs several olfactory subsystems situated at characteristic locations in the nasal cavity to detect and report on different classes of odors. These olfactory subsystems use different neuronal signal transduction pathways, receptor expression repertoires, and axonal projection targets. The Grueneberg Ganglion (GG) is a newly-appreciated olfactory subsystem with receptor neurons located just inside of the nostrils that project axons to a unique domain of interconnected glomeruli in the caudal olfactory bulb. It is not well-understood how the GG relates to other olfactory subsystems in contributing to the olfactory sense. Furthermore, the range of chemoreceptors and the signal transduction cascade utilized by the GG have remained mysterious. To resolve these unknowns, we explored the molecular relationship between the GG and the GC-D neurons, another olfactory subsystem that innervates similarly-interconnected glomeruli in the same bulbar region. We found that mouse GG neurons express the cGMP-associated signaling proteins phosphodiesterase 2a, cGMP-dependent kinase II, and cyclic nucleotide gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia-localized particulate guanylyl cyclases (pGC-G and pGC-A). The primary cGMP signaling pathway of the GG is shared with the GC-D neurons, unifying their target glomeruli as a unique center of olfactory cGMP signal transduction. However, the distinct chemoreceptor repertoire in the GG suggests that the GG is an independent olfactory subsystem. This subsystem is well-suited to detect a unique set of odors and to mediate behaviors that remained intact in previous olfactory perturbations.

The dynamic and restricted expression of the nieuwkoid gene, combined with its potent dorsalizing activity, suggests that nieuwkoid is an important component in the regionalization of the gastrula organizer, possibly characterizing and mediating an organizer-inducing/Nieuwkoop-center-like activity.

The Grueneberg ganglion is a compact cluster of neurons in the rostral nasal vestibule once thought to be a component of the terminal nerve, a non-sensory nerve that does not innervate the olfactory bulb. Its strong expression of olfactory marker protein, a pan-olfactory marker, in mice led us to re-examine this conclusion. Here, we demonstrate that the Grueneberg ganglion projects axons from the nasal vestibule, along the septum, through the cribriform plate and onto the olfactory necklace domain of the olfactory bulbs where it forms glomeruli. Its expression of olfactory marker protein, combined with its direct wiring to the olfactory bulb, strongly suggest that the Grueneberg ganglion is a component of the olfactory pathway.

Dorsoventral specification of the zebrafish gastrula is governed by the functions of the dorsal shield, a region of the embryo functionally analogous to the amphibian Spemann organizer. We report that the bozozok locus encodes the transcription factor nieuwkoid/dharma, a homeobox gene with non-cell-autonomous organizer-inducing activity. The nieuwkoid/dharma gene is expressed prior to the onset of gastrulation in a restricted region of an extraembryonic tissue, the yolk syncytial layer, that directly underlies the presumptive organizer cells. A single base-pair substitution in the nieuwkoid/dharma gene results in a premature stop codon in boz(m168) mutants, leading to the generation of a truncated protein product which lacks the homeodomain and fails to induce a functional organizer in misexpression assays. Embryos homozygous for the boz(m168) mutation exhibit impaired dorsal shield specification often leading to the loss of shield derivatives, such as prechordal plate in the anterior and notochord in the posterior, along the entire anteroposterior axis. Furthermore, boz homozygotes feature a loss of neural fates anterior to the midbrain/hindbrain boundary. Characterization of homozygous mutant embryos using molecular markers indicates that the boz ventralized phenotype may be due, in part, to the derepression of a secreted antagonizer of dorsal fates, zbmp2b, on the dorsal side of the embryo prior to the onset of gastrulation. Furthermore, ectopic expression of nieuwkoid/dharma RNA is sufficient to lead to the down regulation of zbmp2b expression in the pregastrula. Based on these results, we propose that gastrula organizer specification requires the Nieuwkoop center-like activity mediated by the nieuwkoid/dharma/bozozok homeobox gene and that this activity reveals the role of a much earlier than previously suspected inhibition of ventral determinants prior to dorsal shield formation.

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

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