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Articles by James Sharpe in JoVE

 JoVE Clinical and Translational Medicine

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University


JoVE 50238

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

Other articles by James Sharpe on PubMed

Adaptations and Deficits in the Vestibulo-ocular Reflex After Sixth Nerve Palsy

The effects of paralytic strabismus on the vestibulo-ocular reflex (VOR) have not been systematically investigated in humans. The purpose of this study was to analyze the VOR in patients with unilateral peripheral sixth nerve palsy.

Adaptive Neural Mechanism for Listing's Law Revealed in Patients with Sixth Nerve Palsy

During fixation and saccades, human eye movements obey Listing's law, which specifies the torsional eye position for each combination of horizontal and vertical eye positions. To study the mechanisms that implement Listing's law, the authors measured whether the law was violated in peripheral and central unilateral sixth nerve palsy.

Adaptations and Deficits in the Vestibulo-ocular Reflex After Third Nerve Palsy

To analyze the vestibulo-ocular reflex (VOR) in patients with unilateral peripheral third nerve palsy.

Midbrain Disorders of Vertical Gaze: a Quantitative Re-evaluation

The mesodiencephalic junction is the site of the prenuclear control of vertical eye motion. We measured vertical saccades, smooth pursuit (SP), the vertical vestibulo-ocular reflex (VOR), and its interactions with vision during active head motion in 21 patients with midbrain lesions causing palsy of vertical saccades, upward, downward, or in both directions. Most patients with limited slow or slowed saccades in one direction on clinical examination had slowed saccades in the opposed direction. SP gain was decreased in both directions in most patients, and decreased upward or downward in few. VOR gain was subnormal in both directions in many patients, and upward only in one; phase lead of the VOR was recorded in 33% of them. Subnormal SP and VOR gains were often dissociated. Visually enhanced VOR gains were subnormal in both directions in many patients. Cancellation of the VOR was impaired in many patients, both upward and downward in most and upward in few patients. Gaze (eye plus head) tracking gain was subnormal in 29% of patients. Defective SP and defective cancellation of the VOR during head free tracking were often dissociated. We conclude that VOR and SP gains are usually subnormal in patients with paresis of vertical saccades. Impairment of pursuit and the VOR are often dissociated. Phase lead of the VOR implicates damage to velocity-to-position neural integrator for vertical eye motion. These associations and dissociations of impaired vertical eye motion signify discrete structural and functional effects of supranuclear midbrain damage that are undetected by examination of saccades.

Implementation of Listing's Law in Patients with Unilateral Sixth Nerve Palsy

Hyperdeviation and Static Ocular Counterroll in Unilateral Abducens Nerve Palsy

The Vertical Vestibulo-ocular Reflex, and Visual-vestibular Interaction During Active Head Motion

Optical Projection Tomography As a Tool for 3D Microscopy and Gene Expression Studies

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.

Adaptive Neural Mechanism for Listing's Law Revealed in Patients with Fourth Nerve Palsy

During fixation and saccades, human eye movements obey Listing's law, which specifies the eye's torsional angle as a function of its horizontal and vertical position. Torsion of the eye is in part controlled by the fourth nerve. This study investigates whether the brain adapts to defective torsional control after fourth nerve palsy.

Vertical Misalignment in Unilateral Sixth Nerve Palsy

To detect and determine the magnitude of vertical deviation in patients with unilateral sixth nerve palsy.

The Vestibulo-ocular Reflex in Fourth Nerve Palsy: Deficits and Adaptation

The effects of fourth nerve palsy on the vestibulo-ocular reflex (VOR) had not been systematically investigated. We used the magnetic scleral search coil technique to study the VOR in patients with unilateral fourth nerve palsy during sinusoidal head rotations in yaw, pitch and roll at different frequencies. In darkness, VOR gains are reduced during incyclotorsion, depression and abduction of the paretic eye, as anticipated from paresis of the superior oblique muscle. VOR gains during excyclotorsion, elevation and adduction of the paretic eye are also reduced, whereas gains in the non-paretic eye remain normal, indicating a selective adjustment of innervation to the paretic eye. In light, torsional visually enhanced VOR (VVOR) gains in the paretic eye remain reduced; however, visual input increases vertical and horizontal VVOR gains to normal in the paretic eye, without a conjugate increase in VVOR gains in the non-paretic eye, providing further evidence of selective adaptation in the paretic eye. Motions of the eyes after fourth nerve palsy exemplify monocular adaptation of the VOR, in response to peripheral neuromuscular deficits.

Optical Projection Tomography As a New Tool for Studying Embryo Anatomy

Optical projection tomography (OPT) is a new technique for three-dimensional (3D) imaging of small biological tissues. It is particularly useful for reconstructing vertebrate embryos and for examining the 3D anatomy of developing organs. The advantages of this technique over previous methods will be explained: in particular, its ability to image at a higher resolution than magnetic resonance imaging (MRI), while at the same time being able to image specimens much larger than those possible using confocal laser-scanning microscopy. Being an optical technique, OPT is also able to take advantage of the many coloured and fluorescent dyes which have been developed for tissue-specific or gene-specific staining. This becomes particularly important for the visualization of the 3D shapes of specific organs and tissues as it allows the computer to automatically determine the outline of the desired structure.

What's Up, Doc? Altered Perception of the Haptic, Postural, and Visual Vertical

Adaptations and Deficits in the Vestibulo-ocular Reflex After Peripheral Ocular Motor Palsies

Palsy of a nerve might be expected to lower vestibulo-ocular reflex (VOR) responses in its fields of motion, but effects of peripheral neuromuscular disease were unknown. We recorded the VOR during sinusoidal head rotations in yaw, pitch, and roll at 0.5-2 Hz and static torsional gain in 43 patients with unilateral nerve palsies. Sixth nerve palsy (n = 21) reduced both abduction and adduction VOR gains in darkness. In light, horizontal visually enhanced VOR (VVOR) gains were normal in moderate and mild palsy. In severe palsy, horizontal VVOR gains remained low in the paretic eye when it was fixating, whereas gains in the nonparetic eye became higher than normal. Third nerve palsy (n = 10) decreased VOR and VVOR gains during abduction, adduction, elevation, depression, extorsion, and intorsion. Fourth nerve palsy (n = 13) reduced VOR gains of the paretic eye during intorsion, extorsion, elevation, depression, abduction, and adduction, but in light vertical and horizontal VVOR gains were normal. In the nonparetic eye, all gains were normal. Reduced VOR gains in the direction of paretic muscles and also in the direction of their antagonists, together with normal gains in the nonparetic eye, indicate a selective adjustment to the antagonists of paretic muscles. Increase of VVOR gains to normal in the paretic eye, when used for fixation, without conjugate increase in gains in the occluded nonparetic eye, provides further evidence of selective adaptation for the paretic eye. Motions of the eyes after nerve palsies indicate monocular VOR adaptation in three dimensions.

EMAP and EMAGE: a Framework for Understanding Spatially Organized Data

The Edinburgh MouseAtlas Project (EMAP) is a time-series of mouse-embryo volumetric models. The models provide a context-free spatial framework onto which structural interpretations and experimental data can be mapped. This enables collation, comparison, and query of complex spatial patterns with respect to each other and with respect to known or hypothesized structure. The atlas also includes a time-dependent anatomical ontology and mapping between the ontology and the spatial models in the form of delineated anatomical regions or tissues. The models provide a natural, graphical context for browsing and visualizing complex data. The Edinburgh Mouse Atlas Gene-Expression Database (EMAGE) is one of the first applications of the EMAP framework and provides a spatially mapped gene-expression database with associated tools for data mapping, submission, and query. In this article, we describe the underlying principles of the Atlas and the gene-expression database, and provide a practical introduction to the use of the EMAP and EMAGE tools, including use of new techniques for whole body gene-expression data capture and mapping.

Widespread Tangential Dispersion and Extensive Cell Death During Early Neurogenesis in the Mouse Neocortex

The development of the mammalian neocortex requires radial and tangential migration of cells. Radial migration of differentiated neurons from the ventricular zone (VZ) is well established. It is hypothesised that an earlier phase of tangential migration of mitotically active cells lays down a widespread periodically spaced set of progenitors that generate radial arrays of postmitotic neurons. We use a transgenic cell lineage marker to label and observe the behaviour of progenitors before and during the early stages of neurogenesis. Using optical projection tomography (OPT), we show that individual progenitor cells generate many radially arrayed columns of periodically spaced cells. Column positions indicate the paths taken by these progenitor cells as they migrate, often over long distances, through the proliferative zone. Clonally related cells can be distributed in both hemispheres, suggesting progenitor cells cross the midline in the anterior neural plate. We observe a dramatic and rapid decline in the number of labelled clones after E13.5, indicating that there is extensive cell death at this time.

Progressive Ataxia and Palatal Tremor (PAPT): Clinical and MRI Assessment with Review of Palatal Tremors

Palatal tremor has been subdivided into essential (EPT) and symptomatic palatal tremor (SPT). A subgroup of the SPT form has a syndrome of progressive ataxia and palatal tremor (PAPT). Published details of cases of PAPT are sparse and the disorder appears heterogeneous. We present clinical and MRI features of six patients with sporadic PAPT who attended The University Health Network between 1991 and 2002. Eye movements were recorded using a magnetic search coil technique. We review previously reported cases of PAPT from the English language literature and relate this disorder to EPT and SPT. PAPT may be divided into sporadic and familial forms. We identified 22 other prior reported cases of sporadic PAPT. Sporadic PAPT is a subtype of SPT in which progressive cerebellar degeneration is the most symptomatic feature. A combination of vertical nystagmus and palatal tremor was found in one of our cases. Internuclear ophthalmoplegia, a new finding, was present in two of our patients and indicated additional brainstem dysfunction. Inferior olivary high signal abnormalities were present on MRI in all of our cases. The cause of sporadic PAPT remains uncertain. In some previous reports of sporadic PAPT, the combination of brainstem or pontine atrophy, parkinsonism, autonomic dysfunction or corticospinal tract abnormalities suggests a diagnosis of multiple system atrophy, although pathological verification is lacking. Familial PAPT is associated with marked brainstem and cervical cord atrophy with corticospinal tract findings, but the typical olivary MRI abnormalities have not been reported. A substitution in the glial fibrillary acidic protein (GFAP) gene has been described in a family with PAPT, raising the possibility of Alexander's disease. One other familial syndrome of PAPT, termed 'dark dentate disease', has also been reported. PAPT is a subgroup of SPT in which ataxia progresses and is not usually the result of a monophasic illness. Eye movement abnormalities suggest a disorder of both the cerebellum and brainstem. Familial PAPT differs from sporadic PAPT in having marked atrophy of cervical cord and brainstem with corticospinal signs but without hypertrophic olivary appearance on MRI.

Effect of Aging on Visual Shape Distortion

Individuals experience a visual illusion created by shape interaction: when two shapes are presented successively and briefly, the form of the second (test) shape appears distorted due to the form of the first (prime) shape; this shape interaction is called the shape distortion effect. While age-related deterioration in performance is found in various aspects of visual perception, the effect of aging on the shape distortion effect has not been evaluated.

Optical Projection Tomography

Optical projection tomography is a new approach for three-dimensional (3-D) imaging of small biological specimens. It fills an imaging gap between MRI and confocal microscopy, being most suited to specimens that are from 1 to 10 mm across. The tomographic principles of optical projection tomography (OPT) are explained, its most important applications in biomedical research explored, and comparisons drawn of its pros and cons compared to a number of alternative imaging technologies.

3 Dimensional Modelling of Early Human Brain Development Using Optical Projection Tomography

As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped.

Recovery of Peripheral Versus Central Nerves Identified by Saccadic Velocity After Abducens Neuropathy

The abducens is the motor nerve with the most substantial course, both within and outside the brain and it innervates only one muscle. Sixth nerve palsy affords an opportunity to compare recovery after central versus peripheral nerve damage by assessing the dynamics of abduction. Horizontal saccade peak velocities and durations in 14 patients with unilateral peripheral sixth nerve palsies (5 acute, 9 chronic) are compared with those in 5 patients with central sixth nerve palsies (2 acute, 3 chronic) and with those in 10 normal subjects. Acutely, abducting saccades in the paretic eye were slow in both central and peripheral palsies, as anticipated from weakness of the lateral rectus muscle. In chronic central palsies, abducting saccadic velocities remained reduced, but in chronic peripheral palsies, they increased to normal within the limited range of excursion. The chronically damaged peripheral nerve behaves like a high-pass filter in transmitting phasic velocity commands, whereas tonic position commands remain defective, accounting for limited abduction but normal velocities within the range of duction. In chronic central (fascicular) palsies, saccade velocities remain reduced. Impaired conduction from damage to central myelin or axons is more persistent in central palsies, consistent with limited regeneration within the brain. Recording of saccade velocities may aid the distinction of fascicular from peripheral palsies. Saccade speed is repaired in peripheral palsies, probably by remyelination, and perhaps also by central monocular adaptation of innervation selectively to the paretic eye in order to drive both eyes rapidly and simultaneously to a target in the paretic field of motion.

Cerebellar Skew Deviation and the Torsional Vestibuloocular Reflex

Skew deviation is typically caused by brainstem damage, and has not been identified with focal cerebellar lesions. This vertical strabismus has been attributed to asymmetric disruption of vestibuloocular reflex (VOR) projections from otolithic receptors of the utricle to ocular motoneurons, but asymmetry of the utriculo-ocular counter-roll reflex has not been detected.

3D Modelling, Gene Expression Mapping and Post-mapping Image Analysis in the Developing Human Brain

As human brain development proceeds, there are complex changes in size and shape, most notably in the developing forebrain. Molecular technologies enable us to characterise the gene expression patterns that underlie these changes. To interpret these patterns the location of expression must be identified and, often, gene expression patterns compared for several genes or across several developmental stages. To facilitate interpretation we have generated a set of three-dimensional models using a recently developed technique, optical projection tomography. The models act as a framework onto which gene expression patterns are mapped and anatomical domains identified using custom-designed software, MAPaint. Here, we demonstrate their use to compare forebrain development at two embryonic stages (Carnegie stages 18 and 21; 44 and 52 days post conception, respectively) and as a means of recording, storing and visualising gene expression data for three example genes EMX1, EMX2 and OTX2. Anatomical domains were also mapped to the models and the comparison of gene expression and anatomical data is demonstrated at Carnegie stage 21. The three-dimensional models and sophisticated software facilitate the analysis and visualisation of morphological changes and gene expression patterns during early brain development and can be applied to the development of other complex structures.

Correction of Artefacts in Optical Projection Tomography

A new imaging technique called optical projection tomography (OPT), essentially an optical version of x-ray computed tomography (CT), provides molecular specificity, cellular resolution and larger specimen coverage ( approximately 1 cubic centimetre) than was previously possible with other imaging techniques. It is ideally suited to gene expression studies in small animals. Reconstructed OPT images demonstrate several artefacts which reduce the overall image quality. In this paper, we describe methods to prevent smear artefacts due to illumination intensity fluctuation, ring artefacts due to CCD pixel sensitivity variation and a new 'detector edge' artefact caused by non-zero background signal. We also present an automated method to align the position of the rotational axis during image reconstruction. Finally, we propose a method to eliminate bowl artefacts due to projection truncation using a lower resolution OPT scan of the same specimen. This solution also provides OPT with the ability to obtain a high-resolution reconstruction from a region of interest of a specimen that is larger than the field of view. Implementation of these corrections and modifications increases the accuracy of the OPT imaging technique and extends its capabilities to obtain higher resolution data from within a whole specimen.

Saccades in Children

Saccades are necessary for optimal vision. Little is known about saccades in children. We recorded saccades using an infrared eye tracker in 39 children, aged 8-19 years. Participants made saccades to visual targets that stepped 10 degrees or 15 degrees horizontally and 5 degrees or 10 degrees vertically at unpredictable time intervals. Saccadic latency decreased significantly with increasing age, while saccadic gain and peak velocity did not vary with age. Saccadic gains and peak velocities in children are similar to reported adult values. This implies maturity of the neural circuits responsible for making saccades accurate and fast. Saccade latency decreases as the brain matures.

Smooth Pursuit Eye Movements in Children

Smooth pursuit eye movements consists of slow eye movements that approximate the velocity of the eyes to that of a small moving target, so that target image is kept at or near the fovea. Little information on smooth pursuit is available in children. We used an infrared eye tracker to record smooth pursuit in 38 typically developing children, aged 8-19 years. Participants followed a visual target moving sinusoidally at +/-10 degrees amplitude, horizontally and vertically at 0.25 or 0.5 Hz. The mean horizontal smooth pursuit gains, the ratio of eye to target velocities, were 0.84 at 0.25 Hz and 0.73 at 0.5 Hz. Mean vertical smooth pursuit gains were 0.68 at 0.25 Hz and 0.45 at 0.5 Hz. Smooth pursuit gains were significantly lower for vertical in comparison to horizontal tracking, and for 0.5 Hz in comparison to 0.25 Hz tracking (P<0.0001). Smooth pursuit gains increased with age (P<0.01, Pearson's correlation tests), with horizontal gains attaining reported adult values by mid adolescence. Vertical gains had large variability among participants. The median phase, the time interval between eye and target velocities, varied between 39 and 86 ms. Phase was not influenced by age. We conclude that smooth pursuit gains are lower in children than gains reported in adults. Vertical pursuit gain is significantly lower than horizontal pursuit gain. Gains improve with age and approach adult values in mid adolescence. Children have larger phases than reported adults values indicating that prediction in the smooth pursuit system is less mature in children.

Cerebellar Vermis Morphology in Children with Spina Bifida and Chiari Type II Malformation

Posterior fossa size and cerebellar weight and volume are reduced in Chiari type II malformation (CII). This is assumed to affect the cerebellum uniformly. We quantified the presumed reduction in vermis size on magnetic resonance imaging (MRI).

Saccade Dynamics in Peripheral Vs Central Sixth Nerve Palsies

To investigate differences between peripheral idiopathic and central sixth nerve palsies from brainstem damage by comparing peak velocities and durations of horizontal saccades.

Visualizing Plant Development and Gene Expression in Three Dimensions Using Optical Projection Tomography

A deeper understanding of the mechanisms that underlie plant growth and development requires quantitative data on three-dimensional (3D) morphology and gene activity at a variety of stages and scales. To address this, we have explored the use of optical projection tomography (OPT) as a method for capturing 3D data from plant specimens. We show that OPT can be conveniently applied to a wide variety of plant material at a range of scales, including seedlings, leaves, flowers, roots, seeds, embryos, and meristems. At the highest resolution, large individual cells can be seen in the context of the surrounding plant structure. For naturally semitransparent structures, such as roots, live 3D imaging using OPT is also possible. 3D domains of gene expression can be visualized using either marker genes, such as beta-glucuronidase, or more directly by whole-mount in situ hybridization. We also describe tools and software that allow the 3D data to be readily quantified and visualized interactively in different ways.

Spleen Versus Pancreas: Strict Control of Organ Interrelationship Revealed by Analyses of Bapx1-/- Mice

During early stages of pancreatic development, the mesenchyme that contributes to the spleen overlies the dorsal pancreatic endoderm. Here, we show that interactions between splenic mesenchyme and pancreas proceed via a highly orchestrated morphogenetic program. Disruption of morphogenesis, as occurs in the Bapx1(Nkx3.2)(-/-) embryo, results in transformation of these tissues into well-organized, ectopic gut-like structures. Bapx1 plays a crucial organizing role effecting position and separation of the spleen and pancreas to prevent this metaplastic transformation. Similar transformations occur in organ cultures employing wild-type pancreatic endoderm and spleen mesenchyme, revealing the developmental plasticity of the pancreas and that precise spatial and temporal control of tissue interactions are required for development of both organs.

Saccadic Adaptation in Children

Saccades are fast-orienting eye movements. Saccadic adaptation, a form of motor learning, is a corrective change in the amplitude of saccades in response to error. The aim of the study was to ascertain whether saccadic adaptation occurs in typically developing children. We recorded saccades with an infrared eye tracker in 39 children, aged 8 to 19 years, at baseline to 12-degree horizontal target steps and after an adaptive task. During the adaptive task, a saccadic hypometric error was induced. This task consisted of 200 12-degree target steps that stepped backward 3 degrees during the initial saccade and without the participants' awareness. The initial saccade triggered the back-step. This paradigm required a corrective reduction of the amplitude of the initial saccades in response to the induced error. Saccadic adaptation was achieved in 26 participants, whose mean saccadic amplitudes decreased by 13% (P < .05). Saccadic adaptation was not influenced by age. We conclude that children as young as 8 years old have established functions of the neural circuits responsible for the motor learning required for saccadic adaptation.

Saccadic Adaptation in Chiari Type II Malformation

Saccadic adaptation corrects errors in saccadic amplitude. Experimentally-induced saccadic adaptation provides a method for studying motor learning. The cerebellum is a major participant in saccadic adaptation. Chiari type II malformation (CII) is a developmental deformity of the cerebellum and brainstem that is associated with spina bifida. We investigated the effects of CII on saccadic adaptation.

Tomographic Molecular Imaging and 3D Quantification Within Adult Mouse Organs

A convenient technology to quantify three-dimensional (3D) morphological features would have widespread applications in biomedical research. Based on combined improvements in sample preparation, tomographic imaging and computational processing, we present a procedure for high-resolution 3D quantification of structures within intact adult mouse organs. Using the nonobese diabetic (NOD) mouse model, we demonstrate a correlation between total islet beta-cell volume and the onset of type-1 diabetes.

Smooth Ocular Pursuit in Chiari Type II Malformation

Chiari type II malformation (CII) is a congenital anomaly of the cerebellum and brainstem, both important structures for processing smooth ocular pursuit. CII is associated with myelomeningocele and hydrocephalus. We investigated the effects of CII on smooth pursuit (SP) eye movements, and determined the effects of spinal lesion level, number of shunt revisions, nystagmus, and brain dysmorphology on SP. SP was recorded using an infrared eye tracker in 21 participants with CII (11 males, 10 females; age range 8-19y, mean 14y 3mo [SD 3y 2mo]). Thirty-eight healthy children (21 males, 17 females) constituted the comparison group. Participants followed a visual target moving sinusoidally at +/- 10 degrees amplitude, horizontally and vertically at 0.25 or 0.5Hz. SP gains, the ratio of eye to target velocities, were abnormal in the CII group with nystagmus (n= 8). The number of shunt revisions (range 0-10), brain dysmorphology, or spinal lesion level (n= 15 for lower and n= 6 for upper spinal lesion level) did not correlate with SP gains. SP is impaired in children with CII and nystagmus. Abnormal pursuit might be related to the CII dysgenesis or to effects of hydrocephalus. The lack of effect of shunt revisions and abnormal tracking in participants with nystagmus provide evidence that it is related primarily to the cerebellar and brainstem malformation.

Resolution Improvement in Emission Optical Projection Tomography

A new imaging technique called emission optical projection tomography (eOPT), essentially an optical version of single-photon emission computed tomography (SPECT), provides molecular specificity, resolution on the order of microns to tens of microns, and large specimen coverage ( approximately 1 cubic centimetre). It is ideally suited to gene expression studies in embryos. Reconstructed eOPT images suffer from blurring that worsens as the distance from the axis of rotation increases. This blur is caused in part by the defocusing of the lens' point-spread function, which increases with object distance from the focal plane. In this paper, we describe a frequency space filter based on the frequency-distance relationship of sinograms to deconvolve the distance-dependent point-spread function and exclude highly defocused data from the eOPT sinograms prior to reconstruction. The method is shown to reduce the volume at half-maximum of the reconstructed point-spread function to approximately 20% the original, and the volume at 10% maximum to approximately 6% the original. As an illustration, the visibility of fine details in the vasculature of a 9.5 day old mouse embryo is dramatically improved.

FishNet: an Online Database of Zebrafish Anatomy

Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish.

The Vestibulo-ocular Reflex During Active Head Motion in Children and Adolescents

Little information is available on the performance and maturity of the vestibulo-ocular reflex (VOR) in children and adolescents during active head motion as encountered during normal locomotion. We investigated the active VOR performance in children and adolescents to determine its norm and variation with age.

Cell Tracing Reveals a Dorsoventral Lineage Restriction Plane in the Mouse Limb Bud Mesenchyme

Regionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.

Three-dimensional Imaging of Drosophila Melanogaster

The major hindrance to imaging the intact adult Drosophila is that the dark exoskeleton makes it impossible to image through the cuticle. We have overcome this obstacle and describe a method whereby the internal organs of adult Drosophila can be imaged in 3D by bleaching and clearing the adult and then imaging using a technique called optical projection tomography (OPT). The data is displayed as 2D optical sections and also in 3D to provide detail on the shape and structure of the adult anatomy.

Square Wave Jerks in Children and Adolescents

Square wave jerks are involuntary, horizontal, saccadic intrusions that interrupt fixation. Each square wave jerk consists of an initial saccade that moves the fovea away from the intended position of fixation, followed by a second saccade in the opposite direction, which refoveates the fixation position. Square wave jerks reportedly occur in 24-60% of healthy adults. No previous study of square wave jerks in children and adolescents is available. We recorded eye movements using an infrared eye tracker in 38 participants aged 8-19 years while they fixated on a visual target for 1 minute. The frequency of square wave jerks, and the durations, amplitudes, and peak velocities of their saccades, were calculated and correlated with age. Ninety percent of participants had square wave jerks. Their median frequency was 3 per minute (range, 1-18), median duration was 249 milliseconds, the median amplitude of their saccades was 0.81 degrees, and the median peak velocity was 60 degrees/second. No parameter of square wave jerks correlated with age. The prevalence of square wave jerks is high in children and adolescents. This finding may be a feature of the less mature brain, and may reflect an inability to suppress unwanted supranuclear triggers for saccades.

Adaptive Neural Mechanism for Listing's Law Revealed in Patients with Skew Deviation Caused by Brainstem or Cerebellar Lesion

Skew deviation is a vertical strabismus caused by damage to the otolithic-ocular reflex pathway and is associated with abnormal ocular torsion. This study was conducted to determine whether patients with skew deviation show the normal pattern of three-dimensional eye control called Listing's law, which specifies the eye's torsional angle as a function of its horizontal and vertical position.

Localization and Fate of Fgf10-expressing Cells in the Adult Mouse Brain Implicate Fgf10 in Control of Neurogenesis

We used Fgf10-lacZ reporter mice to investigate the distribution and fate of Fgf10-expressing cells in the developing and adult mouse brain. We find that the domain of Fgf10 expression expands post-natally and new niches emerge in the adult brain. Fgf10 is expressed in the adult cerebellum, thalamic, mid- and hindbrain nuclei and hippocampal CA fields, as previously reported in the rat brain. In addition though, we have discovered expression in: the hippocampal dentate gyrus; a discrete trail linking the ventral telencephalon with the olfactory bulbs; ventral ependyma of the third ventricle from where cells appear to disperse into the hypothalamus; and in the pituitary gland. Most Fgf10-expressing cells or their immediate descendants appear immature but a subset differentiates into neurons and glial cells. The manner in which Fgf10 is expressed in these active and quiescent neurogenic niches implicates it in control of neurogenesis and/or conservation of neurogenic potential.

3D Representation of Wnt and Frizzled Gene Expression Patterns in the Mouse Embryo at Embryonic Day 11.5 (Ts19)

Wnt signalling is one of the fundamental cell communication systems operating in the embryo and the collection of 19 Wnt and 10 Frizzled (Fzd) receptor genes (in mouse and human) represent just part of a complex system to be unravelled. Here we present a spatially comprehensive set of data on the 3D distribution of Wnt and Fzd gene expression patterns at a carefully selected single stage of mouse development. Overviews and selected features of the patterns are presented and the full 3D data set, generated by fully described probes, is available to the research community through the Edinburgh Mouse Atlas of Gene Expression. In addition to being comprehensive, the data set has been generated and recorded in a consistent manner to facilitate comparisons between gene expression patterns with the capacity to generate matching virtual sections from the 3D representations for specific studies. Expression patterns in the left forelimb were selected for more detailed comparative description. In addition to confirming the previously published expression of these genes, our whole embryo and limb bud analyses significantly extend the data in terms of details of the patterns and the addition of previously undetected sites of expression. Our focussed analysis of expression domains in the limb, defined by just two gene families, reveals a surprisingly high degree of spatial complexity and underlines the enormous potential for local cellular interactions that exist within an emerging structure. This work also highlights the use of OPT to generate detailed high-quality, spatially complex expression data that is readily comparable between specimens and can be reviewed and reanalysed as required for specific studies. It represents a core set of data that will be extended with additional stages of development and through addition of potentially interacting genes and ultimately other cross-regulatory communication pathways operating in the embryo.

In Vitro Whole-organ Imaging: 4D Quantification of Growing Mouse Limb Buds

Quantitative mapping of the normal tissue dynamics of an entire developing mammalian organ has not been achieved so far but is essential to understand developmental processes and to provide quantitative data for computational modeling. We developed a four-dimensional (4D) imaging technique that can be used to quantitatively image tissue movements and dynamic GFP expression domains in a growing transgenic mouse limb by time-lapse optical projection tomography (OPT).

Dissociated Palsy of Vertical Saccades: Loss of Voluntary and Visually Guided Saccades with Preservation of Reflexive Vestibular Quick Phases

A patient with a diencephalic infarct displayed a persistent palsy of voluntary and visually guided vertical saccades with preserved vertical quick phases of vestibular nystagmus on magnetic search coil oculography. Vertical smooth pursuit had very low velocity in both directions without catch-up saccades. Vertical and torsional vestibulo-ocular reflex gains were normal. Preservation of vertical and torsional quick phases signifies integrity of the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF). This case is the first to provide evidence that disruption of descending cerebral corticofugal pathways to the riMLF with preserved ascending projections from the paramedian pontine reticular formation to the riMLF can cause a dissociated palsy of vertical fast eye movements.

Ocular Motor Nerve Palsies: Implications for Diagnosis and Mechanisms of Repair

Measurements of the dynamics of the eyes in ocular motor nerve palsies may aid diagnosis, characterize peripheral and central palsies, and reveal adaptive properties of the brain. Saccadic and vestibulo-ocular reflex (VOR) functions of patients with peripheral and central sixth, and peripheral third and fourth nerve palsies were studied by three dimensional magnetic field search coil oculography. Combined third and fourth cranial nerve microvascular ischaemic palsy in diabetes mellitus produced low ratios of intorsion to adduction amplitudes. Presumed isolated third nerve palsy caused higher ratios of adduction to intorsion and violations of Listing's law. The VOR in third, fourth, and sixth nerve palsies reveals adaptive equilibration of the action of paretic agonist and their non-paretic antagonist muscles in violation of Hering's law during head motion. Saccadic speeds in the field of paretic agonists are repaired in chronic peripheral palsies despite limited ductions, but remain reduced in central palsies. Limited intorsion with third nerve palsy is attributed to concurrent fourth nerve ischaemia in the distribution of the inferolateral trunk of the intracavernous carotid artery. Adaptive repair of the VOR after ocular motor nerve palsies reduces asymmetric retinal image slip and binocular disparity, and repair of saccadic velocity drives both eyes rapidly and simultaneously into the paretic field of motion.

The Vestibulo-ocular Reflex During Active Head Motion in Chiari II Malformation

Chiari type II malformation (CII) is a developmental anomaly of the cerebellum and brainstem, which are important structures for processing the vestibulo-ocular reflex (VOR). We investigated the effects of the deformity of CII on the angular VOR during active head motion.

Neurophysiology and Neuroanatomy of Smooth Pursuit: Lesion Studies

Smooth pursuit impairment is recognized clinically by the presence of saccadic tracking of a small object and quantified by reduction in pursuit gain, the ratio of smooth eye movement velocity to the velocity of a foveal target. Correlation of the site of brain lesions, identified by imaging or neuropathological examination, with defective smooth pursuit determines brain structures that are necessary for smooth pursuit. Paretic, low gain, pursuit occurs toward the side of lesions at the junction of the parietal, occipital and temporal lobes (area V5), the frontal eye field and their subcortical projections, including the posterior limb of the internal capsule, the midbrain and the basal pontine nuclei. Paresis of ipsiversive pursuit also results from damage to the ventral paraflocculus and caudal vermis of the cerebellum. Paresis of contraversive pursuit is a feature of damage to the lateral medulla. Retinotopic pursuit paresis consists of low gain pursuit in the visual hemifield contralateral to damage to the optic radiation, striate cortex or area V5. Craniotopic paresis of smooth pursuit consists of impaired smooth eye movement generation contralateral to the orbital midposition after acute unilateral frontal or parietal lobe damage. Omnidirectional saccadic pursuit is a most sensitive sign of bilateral or diffuse cerebral, cerebellar or brainstem disease. The anatomical and physiological bases of defective smooth pursuit are discussed here in the context of the effects of lesion in the human brain.

High-resolution Three-dimensional Imaging of Islet-infiltrate Interactions Based on Optical Projection Tomography Assessments of the Intact Adult Mouse Pancreas

A predicament when assessing the mechanisms underlying the pathogenesis of type-1 diabetes (T1D) has been to maintain simultaneous global and regional information on the loss of insulin-cell mass and the progression of insulitis. We present a procedure for high-resolution 3-D analyses of regions of interest (ROIs), defined on the basis of global assessments of the 3-D distribution, size, and shape of molecularly labeled structures within the full volume of the intact mouse pancreas. We apply a refined protocol for optical projection tomography (OPT)-aided whole pancreas imaging in combination with confocal laser scanning microscopy of site-directed pancreatic microbiopsies. As such, the methodology provides a useful tool for detailed cellular and molecular assessments of the autoimmune insulitis in T1D. It is anticipated that the same approach could be applied to other areas of research where 3-D molecular distributions of both global and regional character is required.

Fluorescence Lifetime Optical Projection Tomography

We describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono-exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta.

Evidence That Fgf10 Contributes to the Skeletal and Visceral Defects of an Apert Syndrome Mouse Model

Apert syndrome (AS) is a severe congenital disease caused by mutations in fibroblast growth factor receptor-2 (FGFR2), and characterised by craniofacial, limb, visceral, and neural abnormalities. AS-type FGFR2 molecules exert a gain-of-function effect in a ligand-dependent manner, but the causative FGFs and their relative contribution to each of the abnormalities observed in AS remains unknown. We have generated mice that harbour an AS mutation but are deficient in or heterozygous for Fgf10. The genetic knockdown of Fgf10 can rescue the skeletal as well as some of the visceral defects observed in this AS model, and restore a near normal level of FgfR2 signaling involving an apparent switch between ERK(p44/p42) and p38 phosphorylation. Surprisingly, it can also yield de novo cleft palate and blind colon in a subset of the compound mutants. These findings strongly suggest that Fgf10 contributes to AS-like pathologies and highlight a complexity of Fgf10 function in different tissues.

Visual Fixation in Chiari Type II Malformation

Chiari type II malformation is a congenital deformity of the hindbrain. Square wave jerks are horizontal involuntary saccades that interrupt fixation. Cerebellar disorders may be associated with frequent square wave jerks or saccadic oscillations such as ocular flutter. The effects of Chiari type II malformation on visual fixation are unknown. We recorded eye movements using an eye tracker in 21 participants with Chiari type II malformation, aged 8 to 19 years while they fixated a target for 1 minute. Thirty-eight age-matched healthy participants served as controls. Square wave jerks' parameters were similar in the 2 groups. Saccadic oscillations were not seen. Chiari type II malformation is not associated with pathological square wave jerks or abnormal saccadic oscillations. The congenital nature of this deformity may permit compensation that preserves stable visual fixation. Alternatively, the deformity of Chiari type II malformation may spare parts of the cerebellum that usually cause fixation instability when damaged.

Gene Expression Analysis of Canonical Wnt Pathway Transcriptional Regulators During Early Morphogenesis of the Facial Region in the Mouse Embryo

Structures and features of the face, throat and neck are formed from a series of branchial arches that grow out along the ventrolateral aspect of the embryonic head. Multiple signalling pathways have been implicated in patterning interactions that lead to species-specific growth and differentiation within the branchial region that sculpt these features. A direct role for Wnt signalling in particular has been shown. The spatial and temporal distribution of Wnt pathway components contributes to the operation of the signalling system. We present the precise distribution of gene expression of canonical Wnt pathway transcriptional regulators, Tcf1, Lef1, Tcf3, Tcf4 and beta-catenin between embryonic day (E) 9.5 and 11.5. In situ hybridization combined with Optical Projection Tomography was used to record and compare distribution of transcripts in 3D within the developing branchial arches. This shows widespread yet very specific expression of the gene set indicating that all genes contribute to proper patterning of the region. Tcf1 and Lef1 are more prominent in rostral arches, particularly at later ages, and Tcf3 and Tcf4 are in general expressed more deeply (medial/endodermal aspect) in the arches than Tcf1 and Lef1. Comparison with Wnt canonical pathway readout patterns shows that the relationship between the expression of individual transcription factors and activation of the pathway is not simple, indicating complexity and flexibility in the signalling system.

Genetic Background Influences Embryonic Lethality and the Occurrence of Neural Tube Defects in Men1 Null Mice: Relevance to Genetic Modifiers

Germline mutations of the multiple endocrine neoplasia type 1 (MEN1) gene cause parathyroid, pancreatic and pituitary tumours in man. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP) and the same MEN1 mutations, in different families, can cause either FIHP or MEN1. This suggests a role for genetic background and modifier genes in altering the expression of a mutation. We investigated the effects of genetic background on the phenotype of embryonic lethality that occurs in a mouse model for MEN1. Men1(+/-) mice were backcrossed to generate C57BL/6 and 129S6/SvEv incipient congenic strains, and used to obtain homozygous Men1(-/-) mice. No viable Men1(-/-) mice were obtained. The analysis of 411 live embryos obtained at 9.5-16.5 days post-coitum (dpc) revealed that significant deviations from the expected Mendelian 1:2:1 genotype ratio were first observed at 12.5 and 14.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively (P<0.05). Moreover, live Men1(-/-) embryos were absent by 13.5 and 15.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively thereby indicating an earlier lethality by 2 days in the 129S6/SvEv strain (P<0.01). Men1(-/-) embryos had macroscopic haemorrhages, and histology and optical projection tomography revealed them to have internal haemorrhages, myocardial hypotrophy, pericardial effusion, hepatic abnormalities and neural tube defects. The neural tube defects occurred exclusively in 129S6/SvEv embryos (21 vs 0%, P<0.01). Thus, our findings demonstrate the importance of genetic background in influencing the phenotypes of embryonic lethality and neural tube defects in Men1(-/-) mice, and implicate a role for genetic modifiers.

The Cerebellar Dysplasia of Chiari II Malformation As Revealed by Eye Movements

Chiari type II malformation (CII) is a developmental deformity of the hindbrain. We have previously reported that many patients with CII have impaired smooth pursuit, while few make inaccurate saccades or have an abnormal vestibuloocular reflex. In contrast, saccadic adaptation and visual fixation are normal. In this report, we correlate results from several eye movement studies with neuroimaging in CII. We present a model for structural changes within the cerebellum in CII.

Live Optical Projection Tomography

Optical projection tomography (OPT) is a technology ideally suited for imaging embryonic organs. We emphasize here recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse OPT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further developed by merging state-of-the-art mouse embryo culture with the OPT system. We discuss the technological challenges that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.

Live Optical Projection Tomography

Optical projection tomography (OPT) is a technology ideally suited for imaging embryonic organs. We emphasize here recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse OPT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further developed by merging state-of-the-art mouse embryo culture with the OPT system. We discuss the technological challenges that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.

Quantification and Three-dimensional Imaging of the Insulitis-induced Destruction of Beta-cells in Murine Type 1 Diabetes

The aim of this study was to refine the information regarding the quantitative and spatial dynamics of infiltrating lymphocytes and remaining beta-cell volume during the progression of type 1 diabetes in the nonobese diabetic (NOD) mouse model of the disease.

Clonal Analysis in Mice Underlines the Importance of Rhombomeric Boundaries in Cell Movement Restriction During Hindbrain Segmentation

Boundaries that prevent cell movement allow groups of cells to maintain their identity and follow independent developmental trajectories without the need for ongoing instructive signals from surrounding tissues. This is the case of vertebrate rhombomeric boundaries. Analysis in the developing chick hindbrain provided the first evidence that rhombomeres are units of cell lineage. The appearance of morphologically visible rhombomeres requires the segment restricted expression of a series of transcription factors, which position the boundaries and prefigure where morphological boundaries will be established. When the boundaries are established, when the cells are committed to a particular rhombomere and how they are organized within the hindbrain are important questions to our understanding of developmental regionalization.

Scapula Development is Governed by Genetic Interactions of Pbx1 with Its Family Members and with Emx2 Via Their Cooperative Control of Alx1

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Although the vertebrate limb bud has been studied for decades as a model system for spatial pattern formation and cell specification, the cellular basis of its distally oriented elongation has been a relatively neglected topic by comparison. The conventional view is that a gradient of isotropic proliferation exists along the limb, with high proliferation rates at the distal tip and lower rates towards the body, and that this gradient is the driving force behind outgrowth. Here we test this hypothesis by combining quantitative empirical data sets with computer modelling to assess the potential role of spatially controlled proliferation rates in the process of directional limb bud outgrowth. In particular, we generate two new empirical data sets for the mouse hind limb--a numerical description of shape change and a quantitative 3D map of cell cycle times--and combine these with a new 3D finite element model of tissue growth. By developing a parameter optimization approach (which explores spatial patterns of tissue growth) our computer simulations reveal that the observed distribution of proliferation rates plays no significant role in controlling the distally extending limb shape, and suggests that directional cell activities are likely to be the driving force behind limb bud outgrowth. This theoretical prediction prompted us to search for evidence of directional cell orientations in the limb bud mesenchyme, and we thus discovered a striking highly branched and extended cell shape composed of dynamically extending and retracting filopodia, a distally oriented bias in Golgi position, and also a bias in the orientation of cell division. We therefore provide both theoretical and empirical evidence that limb bud elongation is achieved by directional cell activities, rather than a PD gradient of proliferation rates.

Mechanobiology of Embryonic Skeletal Development: Insights from Animal Models

A range of clinical conditions in which fetal movement is reduced or prevented can have a severe effect on skeletal development. Animal models have been instrumental to our understanding of the interplay between mechanical forces and skeletal development, particularly the mouse and the chick model systems. In the chick, the most commonly used means of altering the mechanical environment is by pharmaceutical agents which induce paralysis, whereas genetically modified mice with nonfunctional or absent skeletal muscle offer a valuable tool for examining the interplay between muscle forces and skeletogenesis in mammals. This article reviews the body of research on animal models of bone or joint formation in vivo in the presence of an altered or abnormal mechanical environment. In both immobilized chicks and "muscleless limb" mice, a range of effects are seen, such as shorter rudiments with less bone formation, changes in rudiment and joint shape, and abnormal joint cavitation. However, although all bones and synovial joints are affected in immobilized chicks, some rudiments and joints are unaffected in muscleless mice. We propose that extrinsic mechanical forces from movements of the mother or littermates impact on skeletogenesis in mammals, whereas the chick embryo is reliant on intrinsic movement for mechanical stimulation. The insights gained from animal models into the mechanobiology of embryonic skeletal development could provide valuable cues to prospective tissue engineers of cartilage and bone and contribute to new or improved treatments to minimize the impact on skeletal development of reduced movement in utero.

Involvement of the Human Ventrolateral Thalamus in the Control of Visually Guided Saccades

An Atlas of Gene Regulatory Networks Reveals Multiple Three-gene Mechanisms for Interpreting Morphogen Gradients

The interpretation of morphogen gradients is a pivotal concept in developmental biology, and several mechanisms have been proposed to explain how gene regulatory networks (GRNs) achieve concentration-dependent responses. However, the number of different mechanisms that may exist for cells to interpret morphogens, and the importance of design features such as feedback or local cell-cell communication, is unclear. A complete understanding of such systems will require going beyond a case-by-case analysis of real morphogen interpretation mechanisms and mapping out a complete GRN 'design space.' Here, we generate a first atlas of design space for GRNs capable of patterning a homogeneous field of cells into discrete gene expression domains by interpreting a fixed morphogen gradient. We uncover multiple very distinct mechanisms distributed discretely across the atlas, thereby expanding the repertoire of morphogen interpretation network motifs. Analyzing this diverse collection of mechanisms also allows us to predict that local cell-cell communication will rarely be responsible for the basic dose-dependent response of morphogen interpretation networks.

4D Retrospective Lineage Tracing Using SPIM for Zebrafish Organogenesis Studies

A study demonstrating an imaging framework that permits the determination of cell lineages during organogenesis of the posterior lateral line in zebrafish is presented. The combination of Selective Plane Illumination Microscopy and specific fluorescent markers allows retrospective tracking of hair cell progenitors, and hence the derivation of their lineages within the primodium. It is shown that, because of its superior signal-to-noise ratio and lower photo-damaged properties, SPIM can provide significantly higher-quality images than Spinning Disk Confocal technology. This allows accurate 4D lineage tracing for the hair cells over tens of hours of primordium migration and neuromast development.

Ocular Torsion and Vertical Misalignment

This article considers vertical misalignment and torsion of the eyes that arise from disorders of vestibulo-ocular reflex (VOR) pathways.

A Landmark-free Morphometric Staging System for the Mouse Limb Bud

We have created a 2D morphometric analysis of the developing mouse hindlimb bud. This analysis has provided two useful resources for the study of limb development. First, a temporally accurate numerical description of shape changes during normal mouse limb development. Second, a web-based morphometric staging system, which has the advantage of being easy to use, and with a reproducibility of about ±2 hours. It allows users to upload a dorsal-view photo of a limb bud, draw a spline curve and thereby stage the bud within a couple of minutes. We describe how the system is constructed, its robustness to user variation and illustrate one application: the accurate tracking of spatiotemporal dynamics of gene expression patterns.

A Computational Clonal Analysis of the Developing Mouse Limb Bud

A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

Budding Behaviors: Growth of the Limb As a Model of Morphogenesis

Questions regarding morphogenesis have played second fiddle to those pertaining to pattern formation among the limb development set for some time. A recent series of publications has reinvigorated the search for mechanisms by which the limb bud arises, elongates and acquires its peculiar shape. While there are stage-specific variations, the theme that resonates across these studies is that mesoderm and cartilage cells in the limb bud exhibit polarity that drives directional movement and oriented division. Noncanonical Wnt signalling is important for these cell behaviors at all stages of limb development. While the emerging morphogenetic mechanisms underlying limb bud outgrowth are partly analogous to those of other developing structures, insights from the limb have the potential to reveal intriguing new mechanisms by which three dimensional mesoderm changes shape.

Control of Pelvic Girdle Development by Genes of the Pbx Family and Emx2

Genes expressed in the somatopleuric mesoderm, the embryonic domain giving rise to the vertebrate pelvis, appear important for pelvic girdle formation. Among such genes, Pbx family members and Emx2 were found to genetically interact in hindlimb and pectoral girdle formation. Here, we generated compound mutant embryos carrying combinations of mutated alleles for Pbx1, Pbx2, and Pbx3, as well as Pbx1 and Emx2, to examine potential genetic interactions during pelvic development. Indeed, Pbx genes share overlapping functions and Pbx1 and Emx2 genetically interact in pelvic formation. We show that, in compound Pbx1;Pbx2 and Pbx1;Emx2 mutants, pelvic mesenchymal condensation is markedly perturbed, indicative of an upstream control by these homeoproteins. We establish that expression of Tbx15, Prrx1, and Pax1, among other genes involved in the specification and development of select pelvic structures, is altered in our compound mutants. Lastly, we identify potential Pbx1-Emx2-regulated enhancers for Tbx15, Prrx1, and Pax1, using bioinformatics analyses.

N-myc Controls Proliferation, Morphogenesis, and Patterning of the Inner Ear

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27(Kip1) and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

Posterior Reversible Encephalopathy Syndrome Presenting As Balint Syndrome

Balint syndrome is a disorder of inaccurate visually guided saccades, optic ataxia, and simultanagnosia that typically results from bilateral parieto-occipital lesions. Visual perception disturbances in the posterior reversible encephalopathy syndrome (PRES) include hemianopia, visual neglect, and cerebral blindness, but Balint syndrome had not been recognized. We report Balint syndrome associated with PRES in a 37-year-old woman with acute hypertension and systemic lupus erythematosus. Balint syndrome can be an initial presentation of PRES.

Preparation of Mouse Embryos for Optical Projection Tomography Imaging

Optical Projection Tomography of Vertebrate Embryo Development

Two Ways to Use Imaging: Focusing Directly on Mechanism, or Indirectly Via Behaviour?

Recent developments in mesoscopic imaging--imaging at the level of tissues and organs, rather than the subcellular or molecular scale--are proving to be powerful for developmental biology. At the same time, these developments are also helping to emphasize an important distinction between two quite different approaches of how imaging is used. In the more traditional approach, images provide a direct insight into how a systems works-suggesting a mechanism or part of a mechanism. However an alternative approach is gaining ground, in which imaging is used to quantify the behaviour of a system, rather than directly assessing the mechanism. In this case the causal relationships of a system are inferred in a more indirect way-by comparing quantitative measurements with mathematical models of the system in question. Although indirect, this approach is powerful for addressing more complex biological systems--especially multiscale problems. It is tempting to distinguish the latter approach with the label 'quantitative biology', but this term only emphasizes the use of numbers, and therefore obscures the more fundamental difference, which is the powerful but indirect nature of the approach. Here I will discuss the distinction between the two imaging approaches, particularly in the context of recent improvements to tissue-level imaging techniques.

Antisaccade Generation is Impaired After Parietal Lobe Lesions

Antisaccades are directed away from visual targets. Impaired antisaccade generation has been attributed to frontal lobe damage. We studied antisaccades in patients with unilateral focal parietal lobe lesions. Normal subjects (N = 10) instructed to make 10° antisaccades opposite to a 100-ms target flash 10° to the right or left of center made antisaccades in 86.1% of trials. Patients (N = 13) made antisaccades contraversive to their lesions in 55.4% of trials and 50.5% of ipsiversive trials. In other trials, reflexive saccades occurred toward the target flash. Nine patients with imaged lesions overlapping in parietal lobe white matter showed subnormal antisaccade generation. Antisaccades provide a means of measuring voluntary saccade function of the parietal lobes independent of visual guidance. Impaired suppression of reflexive saccades and generation of antisaccades is attributed to disconnection of parietal lobe from frontal lobe ocular motor areas.

Image Processing Assisted Algorithms for Optical Projection Tomography

Since it was first presented in 2002, optical projection tomography (OPT) has emerged as a powerful tool for the study of biomedical specimen on the mm to cm scale. In this paper, we present computational tools to further improve OPT image acquisition and tomographic reconstruction. More specifically, these methods provide: semi-automatic and precise positioning of a sample at the axis of rotation and a fast and robust algorithm for determination of postalignment values throughout the specimen as compared to existing methods. These tools are easily integrated for use with current commercial OPT scanners and should also be possible to implement in "home made" or experimental setups for OPT imaging. They generally contribute to increase acquisition speed and quality of OPT data and thereby significantly simplify and improve a number of three-dimensional and quantitative OPT based assessments.

Image Formation by Linear and Nonlinear Digital Scanned Light-sheet Fluorescence Microscopy with Gaussian and Bessel Beam Profiles

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Quantitative Measurements in 3-dimensional Datasets of Mouse Lymph Nodes Resolve Organ-wide Functional Dependencies

Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wild-type and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response.

Transfecting RNA Quadruplexes Results in Few Transcriptome Perturbations

Guanine-rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. Previous studies showed that transfecting G-quadruplex DNA oligonucleotides inhibits proliferation in many cancer cell lines and can induce apoptosis. However, little is known about the effects of transfecting RNA quadruplexes. In this study, we transfected a G-quadruplex RNA oligonucleotide (GqRNA) into HEK293T cells and observed that it did not alter cell viability. Subsequent transcriptome expression profiling revealed that only two genes, EGR1 and FOS, were significantly altered in the presence of GqRNA (upregulated 2- to 4-fold). Sequence analysis showed that both genes contained putative quadruplex sequences (PQS) in their 3'-UTRs, immediately adjacent to the stop codons. Transfection of the EGR1 PQS as an RNA oligonucleotide also caused an increase in EGR1 expression. Similar motifs are found in a variety of genomes, but are relatively rare and have been missed by previous annotations. A bioinformatic analysis revealed stop codon-proximal enrichment of such motifs compared with the rest of the 3'-UTR, although these genes were not affected by RNA quadruplex transfection, and their function remains unknown. Overall, transfecting RNA quadruplexes results in relatively few alterations in gene expression.

Hox Genes Regulate Digit Patterning by Controlling the Wavelength of a Turing-type Mechanism

The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.

A Global "imaging'' View on Systems Approaches in Immunology

The immune system exhibits an enormous complexity. High throughput methods such as the "-omic'' technologies generate vast amounts of data that facilitate dissection of immunological processes at ever finer resolution. Using high-resolution data-driven systems analysis, causal relationships between complex molecular processes and particular immunological phenotypes can be constructed. However, processes in tissues, organs, and the organism itself (so-called higher level processes) also control and regulate the molecular (lower level) processes. Reverse systems engineering approaches, which focus on the examination of the structure, dynamics and control of the immune system, can help to understand the construction principles of the immune system. Such integrative mechanistic models can properly describe, explain, and predict the behavior of the immune system in health and disease by combining both higher and lower level processes. Moving from molecular and cellular levels to a multiscale systems understanding requires the development of methodologies that integrate data from different biological levels into multiscale mechanistic models. In particular, 3D imaging techniques and 4D modeling of the spatiotemporal dynamics of immune processes within lymphoid tissues are central for such integrative approaches. Both dynamic and global organ imaging technologies will be instrumental in facilitating comprehensive multiscale systems immunology analyses as discussed in this review.

Genetics of System Biology

Turing Patterns in Development: What About the Horse Part?

For many years Turing patterns-the repetitive patterns which Alan Turing proved could arise from simple diffusing and interacting factors-have remained an interesting theoretical possibility, rather than a central concern of the developmental biology community. Recently however, this has started to change, with an increasing number of studies combining both experimental and theoretical work to reveal how Turing models may underlie a variety of patterning or morphogenetic processes. We review here the recent developments in this field across a wide range of model systems.

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