Gaëlle Recher

Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy.

We performed Second Harmonic Generation (SHG) imaging microscopy of endogeneous myosin-rich and collagen-rich tissues in amphibian and mammals. We determined the relative components of the macroscopic susceptibility tensor chi((2)) from polarization dependence of SHG intensity. The effective orientation angle theta(e) of the harmonophores has been determined for each protein. For myosin we found theta(e) approximately 62 degrees and this value was unchanged during myofibrillogenesis. It was also independent of the animal species (xenopus, dog and human). For collagen we found theta(e) approximately 49 degrees for both type I- and type III- rich tissues. From these results we localized the source of SHG along the single helix of both myosin and collagen.

Dipolar versus octupolar triphenylamine-based fluorescent organic nanoparticles as brilliant one- and two-photon emitters for (bio)imaging.

Two related triphenylamine-based dipolar and octupolar fluorophores are used to prepare aqueous suspensions of fluorescent organic nanoparticles (FONs) via the reprecipitation method. The obtained spherical nanoparticles (30-40 nm in diameter) are fluorescent in aqueous solution (up to 15% fluorescence quantum yield) and exhibit extremely high one- and two-photon brightness, superior to those obtained for quantum dots. Despite the two chromophores showing similar fluorescence in solution, the fluorescence of FONs made from the octupolar derivative is significantly red-shifted compared to that generated by the dipolar FONs. In addition, the maximum two-photon absorption cross section of the FONs made from the octupolar derivative is 55% larger than that of the dipolar derivative FONs. The experimental observations provide evidence that the different molecular shape (rodlike versus three-branched) and charge distribution (dipolar versus octupolar) of the two chromophores strongly affect the packing inside the nanoparticles as well as their spectroscopic properties and colloidal stability in pure water. The use of these FONs as probes for biphotonic in-vivo imaging is investigated on Xenopus laevis tadpoles to test their utilization for angiography. When using FONs made from the octupolar dye, the formation of microagglomerates (2-5 μm scale) is observed in vivo, with subsequent lethal occlusion of the blood vessels. Conversely, the nanoparticles of the dipolar dye allow acute imaging of blood vessels thanks to their suitable size and brightness, while no toxic effect is observed. Such a goal cannot be achieved with the dissolved dye, which permeates the vessel walls.

A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage.

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology.

Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy

We have extensively characterized the sarcomeric SHG signal as a function of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) and we found that the main feature of this signal is a single peak per mature sarcomere (about 85% of all sarcomeres). The remaining (15%) was found to be either double peak per mature sarcomere or mini sarcomeres (half of a sarcomere) using alpha-actinin immuno detection of the Z-band. The mini sarcomeres are often found in region of pitchfork-like SHG pattern. We suggest that double peak SHG pattern could indicate regions of sarcomeric proteolysis whereas pitchfork-like SHG pattern could reveal sarcomeric assembly.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis

Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

Pluripotent state transitions coordinate morphogenesis in mouse and human embryos

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.

Double-band sarcomeric SHG pattern induced by adult skeletal muscles alteration during myofibrils preparation.

To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.

Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles.

SHG angular intensity pattern (SHG-AIP) of healthy and proteolysed muscle tissues are simulated and imaged here for the first time to our knowledge. The role of the spatial distribution of second-order nonlinear emitters on SHG-AIP is highlighted. SHG-AIP with two symmetrical spots is found to be a signature of healthy muscle whereas SHG-AIP with one centered spot in pathological mdx muscle is found to be a signature of myofibrillar disorder. We also show that SHG-AIP provides information on the three-dimensional structural organization of myofibrils in physiological and proteolysed muscle. Our results open an avenue for future studies aimed at unraveling more complex physiological and pathological fibrillar tissues organization.

Skeletal muscle sarcomeric SHG patterns photo-conversion by femtosecond infrared laser

Femtosecond laser at 780 nm excitation wavelength was used to photo-convert the physiological sarcomeric single band (SB) second harmonic generation (SHG) pattern into double band (DB) in Xenopus laevis premetamorphic tail muscles. This photo-conversion was found to be a third order non-linear optical process and was drastically reduced at 940 nm excitation wavelength. This effect was no longer observed in paraformaldehyde fixed muscles and was enhanced by hydrogen peroxide. The action of hydrogen peroxide suggests that reactive oxygen species (ROS) could contribute to this photo-conversion. These results demonstrate that sarcomeric DB SHG pattern is a marker of sarcomere photodamage in xenopus tadpole muscles and highlight the need of being very careful at using two-photon excitation while observing living tissues. Moreover they open new avenues for in situ intravital investigation of oxidative stress effects in muscle dysfunctions and diseases.

Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters

Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopusleavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.