clonal-genetic-tracing-using-confetti-mouse-to-study-mineralized

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Karolinska Institutet

The peripheral nervous system (PNS) is complex and omnipresent. The PNS targets all parts of the body starting from early stages of embryonic development, and in large part, is derived from multipotent migratory neural crest stem cells. Current opinion mostly perceives the PNS as a means of communication and information exchange between the central nervous system, the rest of the body and the environment. Additionally, the PNS is largely associated with autonomic control. Being an "alternative brain" it provides local regulation of processes in organs. However, it has become evident in recent years that in addition to these main canonical functions the PNS possesses a number of other important roles in development and homeostasis of targeted tissues, for instance, in nerve-dependent regeneration. The PNS represents a niche that hosts neural crest-derived peripheral glial cells, or, in other words, neural crest-like multipotent cells throughout the entire body. These multipotent nerve-adjacent cells can be reprogrammed in vivo and play a number of roles from creating pigmentation to controlling regeneration of a limb in amphibians or skin in rodents. In the current review we outline newly emerged, non-canonical functions of the PNS and briefly describe cellular and molecular aspects of these alternative functions. 

Non-canonical functions of the peripheral nerve.

Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

Glial origin of mesenchymal stem cells in a tooth model system.

The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions. However, cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly also high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing.

Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway.

Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

Analysis of neural crest-derived clones reveals novel aspects of facial development.

Spotlight on the Schwann cells during the regeneration.

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

Chrondrocranium, the cartilaginous skull, is one of the major innovations that underlie evolution of the vertebrate head. Control of the induction and shaping of the cartilage is a key for the formation of the facial bones and largely defines facial shape. The appearance of cartilage in the head enabled many new functions such as protection of central nervous system and sensory structures, support of the feeding apparatus and formation of muscle attachment points ensuring faster and coordinated jaw movements. Here we review the evolution of cartilage in the cranial region and discuss shaping of the chondrocranium in different groups of vertebrates.

Evolution and development of the cartilaginous skull: From a lancelet towards a human face.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Scaling and shaping of vertebrate muscles and skeletal elements has always been enigmatic and required an advanced technical level in order to analyse the cell distribution in 3D. In this work, synchrotron X-ray computed microtomography (µCT) and chemical contrasting has been exploited for a quantitative analysis of the 3D-cell distribution in tissues of a developing salamander (Pleurodeles waltl) limb - a key model organism for vertebrate regeneration studies. We mapped the limb muscles, their size and shape as well as the number and density of cells within the extracellular matrix of the developing cartilage. By using tomographic approach, we explored the polarity of the cells in 3D, in relation to the structure of developing joints. We found that the polarity of chondrocytes correlates with the planes in joint surfaces and also changes along the length of the cartilaginous elements. Our approach generates data for the precise computer simulations of muscle-skeletal regeneration using cell dynamics models, which is necessary for the understanding how anisotropic growth results in the precise shapes of skeletal structures.

A quantitative analysis of 3D-cell distribution in regenerating muscle-skeletal system with synchrotron X-ray computed microtomography.

Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor  The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.

Spatiotemporal structure of cell fate decisions in murine neural crest.

Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton.

Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish.

3D imaging approaches based on X-ray microcomputed tomography (microCT) have become increasingly accessible with advancements in methods, instruments and expertise. The synergy of material and life sciences has impacted biomedical research by proposing new tools for investigation. However, data sharing remains challenging as microCT files are usually in the range of gigabytes and require specific and expensive software for rendering and interpretation. Here, we provide an advanced method for visualisation and interpretation of microCT data with small file formats, readable on all operating systems, using freely available Portable Document Format (PDF) software. Our method is based on the conversion of volumetric data into interactive 3D PDF, allowing rotation, movement, magnification and setting modifications of objects, thus providing an intuitive approach to analyse structures in a 3D context. We describe the complete pipeline from data acquisition, data processing and compression, to 3D PDF formatting on an example of craniofacial anatomical morphology in the mouse embryo. Our procedure is widely applicable in biological research and can be used as a framework to analyse volumetric data from any research field relying on 3D rendering and CT-biomedical imaging.

Article	Year
Non-canonical functions of the peripheral nerve. Experimental cell research\| PubMed ID: 24140263	2014
Glial origin of mesenchymal stem cells in a tooth model system. Nature\| PubMed ID: 25079316	2014
Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway. Cell communication and signaling : CCS\| PubMed ID: 25627785	2015
Analysis of neural crest-derived clones reveals novel aspects of facial development. Science advances\| PubMed ID: 27493992	2016
Spotlight on the Schwann cells during the regeneration. Stem cell investigation\| PubMed ID: 27868056	2016
Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage. eLife\| PubMed ID: 28414273	2017
Evolution and development of the cartilaginous skull: From a lancelet towards a human face. Seminars in cell & developmental biology\| PubMed ID: 29248472	2019
Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage. eLife\| PubMed ID: 29897331	2018
A quantitative analysis of 3D-cell distribution in regenerating muscle-skeletal system with synchrotron X-ray computed microtomography. Scientific reports\| PubMed ID: 30237460	2018
Spatiotemporal structure of cell fate decisions in murine neural crest. Science (New York, N.Y.)\| PubMed ID: 31171666	2019
Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31285319	2019
An interactive and intuitive visualisation method for X-ray computed tomography data of biological samples in 3D Portable Document Format. Scientific reports\| PubMed ID: 31624273	2019

Marketa Kaucka

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