Lineage tracing of early organogenesis and liver mesenchymal cells with ultrasound-guided in utero nano-injection

Current methods used to investigate embryo development and alter gene expression in mouse embryos are often time consuming and require large numbers of mice. To circumvent these limitations, we were aiming to develop a flexible and efficient method to investigate embryo development and manipulate gene expression in utero. Two tissues of interest to target in vivo are the ectodermal/ neural compartment, and mesoderm. Neurectoderm gives rise to the brain, spinal cord and peripheral nervous system, among others, while mesoderm gives rise to blood, muscle, and mesenchymal cells, among other cell types.

Mesenchymal cells in liver, including peri-portal fibroblasts, mesothelial cells and hepatic stellate cells (HSCs), play multiple crucial roles in normal liver development, liver regeneration or liver fibrosis when liver is injured. Mesenchymal cells express both neural and mesenchymal markers, therefore both a neural crest (NC) and mesoderm origin have been proposed. The embryonic origin of HSCs is a long-debated topic in this field due to the lack of specific marker genes and potential convergent differentiation from different origins.

To investigate nervous system development, we first hypothesized that by injecting lentivirus into the amniotic cavity (AC) prior to the neural plate closure, the open neural plate should be labeled and therefore label the future brain and spinal cord. In Paper I and II, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to transduce either nervous system with up to 99% efficacy or selectively achieve the expression in specific cell types by using cell-specific MiniPromoters.

In Paper III, the first aim was to develop a method to target mesoderm, and then to apply this technology to investigate liver mesenchymal cells. We hypothesized that exclusive labeling of the mesoderm and its progeny could be achieved by injecting into the exocoelomic cavity (ExC) during gastrulation after the segregation of three germ layers and full establishment of two cavities (AC and ExC), since mesoderm is in contact with the ExC. Therefore, we further adapted ultrasound-guided in utero nano-injection to lineage trace mesoderm descendants by injecting a diverse lentivirus barcode library into the ExC at embryonic day (E)7.5. In parallel, we used NEPTUNE, to target ectoderm/neural crest as well as primitive streak (PS) by injecting into AC at E7.5. Embryos were collected at E9.5 and E10.5 to address early organogenesis and the origin of septum transversum mesenchyme (STM), a transit tissue believed to contribute to the mesenchymal compartments of diverse internal organs. E16.5 livers were collected to resolve the clonal relations between different mesenchymal cells in the liver.

In summary, during my doctoral studies, we developed two new approaches to target embryonic tissues during development. Ultrasound-guided in utero nano-injection is a flexible and efficient tool to elucidate clonal relations among tissues in early mouse embryo development, as well as for gene manipulation. It significantly minimizes both the financial cost and ethical burdens associated with animal research, in the meantime accelerating the progression from hypothesis to in vivo results.

List of scientific papers

I. Mangold, K., Mašek, J.*, He, J.*, Lendahl, U., Fuchs, E., & Andersson, E. R. (2021). Highly efficient manipulation of nervous system gene expression with NEPTUNE. Cell Reports Methods. 1(4):100043. *These authors have equal contribution.
https://doi.org/10.1016/j.crmeth.2021.100043

II. Mangold, K., He, J., Stokman, S., & Andersson, E. R. (2022). Murine Neural Plate Targeting by In Utero Nano-injection (NEPTUNE) at Embryonic Day 7.5. JoVE (Journal of Visualized Experiments). (180):e63148.
https://doi.org/10.3791/63148

III. Jingyan He, Sandra De Haan, Lenka Belicova, Agustin Corbat, Noémi Van Hul, Stefaan Verhulst, Michael Ratz, Leo Van Grunsven, Jonas Frisén, Emma R Andersson. Single cell lineage tracing of ectodermal and mesodermal compartments contributing to organogenesis and liver mesenchymal cells. [Manuscript]