In The Spotlight
We read an interesting paper! Here is what it says…about intravital imaging
Written by Chiara
Long-term imaging of developing embryos has not been easy to accomplish. The different approaches available so far do not allow to combine the use of fluorescent markers with the imaging techniques employed (i.e. ultrasounds and magnetic resonance, MRI), or cannot be used while the embryos are still in the uterus (i.e. extra-uterine culturing of embryos for imaging). Scientists from the Shen lab at Duke University successfully developed a breakthrough technique that allows intravital imaging of mouse embryos from embryonic day (E) 9.5 till birth (Huang et al, Science 2020). The paper came out in April 2020, amidst the COVID19 pandemic, and showcases the main features of the new imaging preparation methodology, as well as different possible applications.
The idea of making a window implant on the abdomen of pregnant mice (dams) to image developing embryos came to Quiang Huang, co-first author of the paper and at the time a pediatric gastrointestinal surgeon visiting the Shen lab from China. The required material seems quite trivial: surgical equipment, a glass coverslip and a 3D printer. Thanks to a 3D-printed frame and clip, you have a perfectly functioning window implant, which you can cover with a glass coverslip for imaging. Since the uterine muscle and the decidua, the modified mucosa lining the uterus during pregnancy would hamper clear vision and imaging of the embryo, they need to be stripped for imaging from E9.5 until E12.5.
There are two different versions of the window: a round, 10mm diameter window to image embryos from E9.5 up to E12.5; an oval, 15mm long window to image embryos from E13.5 until birth. An important feature of this implantable window is that it is also removable, which allows manipulations of the embryo for experimental reasons, or to simply change the window from the round to the oval if the imaging has to continue longer than the thirteenth day of gestation (in fact, from E13.5 on, the embryo gets too big to completely fit under the round window). Moreover, it is necessary to suture the embryo to the abdominal muscle and the latter to the window, in order to avoid motions as a reflection of the dam’s breathing. Interestingly, implanted dams seem to go through gestation without problems, they usually give birth to pups naturally and feed the pups normally, indicating little to no adverse consequences of the presence of the window implant.
The authors showed different examples of how you can use the window for intravital imaging with stereoscopic microscopy and 2-photon microscopy. Imaging can be performed on transgenic embryos, embryos that were in utero electroporated, chimeric embryos, as well as embryos that received adeno associated virus (AAV) injections for gene therapy. It is also possible to visualize fluorescent dyes that were injected in the dam via tail injection and diffused to the embryo.
While it is exciting to see the live imaging of neurotransmission in Thy1-GCaMP6 (expressing the calcium indicator GCaMP6 in neurons) embryos after photoexcitation (see Supplemental Movie S5), there are some limitations to the use of this implantable window that need to be considered.
Firstly, Huang and colleagues applied only one window per dam, which makes the technique quite costly in terms of animals to use. Perhaps it will be possible to improve the surgery in such a way to have two windows implanted at the same time, one per uterine horn, but for now it is possible to image only one embryo per dam. Secondly, one must take into account the viability of embryos. The team at Duke University managed to reach a viability of more than 80% for the embryos developing under the windows, with only a slight weight reduction and no developmental alterations. However, the viability can be reduced when performing additional manipulations of the embryos, such as in utero electroporation or viral injections in the embryos. Moreover, birth rates are higher when the embryos grow under the round window, due to the lack of abdominal contractions during labor. Another observation made by the authors is that different vectors for the overexpression of transgenes show different expression, efficiency and stability, which depends on the specific use of vectors and promoters.
This new method for intravital imaging opens a new window on studying mammalian development in an unprecedented way. The knowledge gathered from these studies will help elucidate the spatiotemporal dynamics of development in the nervous system and in other tissues, and to get direct visual proof of the effects of genetic manipulations and pharmacological treatments on developing embryos.
Written by Chiara Galante; Edited by Radhika Menon. Featured Image: NGC/Design.