James Sharpe

The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis.

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.

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.

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.

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.

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.

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 vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

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. Developmental Dynamics 238:376–385, 2009.

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. Developmental Dynamics 240:1054–1062, 2011.

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.