Ultrasound-guided in utero lentivirus transduction of the murine nervous system to investigate nervous system disorders

Abstract

Neurodevelopment is a highly dynamic process involving hundreds of molecular different cell types. Gain and loss of function studies provide valuable insight into the gene regulatory networks that orchestrate this morphological process. However, current methods to alter gene expression in mice either lack efficacy or consume a lot of time and resources.

In Paper I we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction). This describes a technique, where lentivirus is injected into the amniotic cavity of embryonic day 7.5 mouse embryos in order to transduce the neural plate prior to the onset of neurulation. After determining optimal injection parameters, NEPTUNE achieves widespread infection of the brain and spinal cord, and transduction was stable for at least 6 months. Using cell type specific MiniPromoters, NEPTUNE could be adapted for conditional expression in neurons and glial cells. Finally, knock down of Olig2 recapitulated the phenotype of Olig2-/- embryos, while knock down of Sptbn2 induced neural tube defects, therefore suggesting a novel function of Sptbn2 during neurodevelopment.

In Paper II we prepared a detailed and through protocol to provide all the necessary information that enables reproducibility of the technique. In concordance with the 3Rs we have developed a staging protocol to assess amniotic cavities prior to surgery and determined maximum volumes that can be injected without compromising embryonic survival. We further highlight the flexible usage of NEPTUNE for either assessment of gene function using widespread transduction or for lineage tracing using clonal transduction. Finally, we address what other organs and tissues are targeted with non-conditional NEPTUNE, including the eye, neural crest cells and the peripheral nervous system.

In Paper III we use NEPTUNE to manipulate gene expression in the developing enteric nervous system. Single cell RNA sequencing of E9.5 embryos showed that NEPTUNE targets neural crest cells and neural crest derived tissues, for example cardiomyocytes, as well as enteric progenitors. Non-conditional NEPTUNE showed even transduction of both, neuronal and glial progenitors in the stomach, small and large intestine. By applying NEPTUNE in Baf53b-Cre transgenic mouse line, we achieved conditional targeting of neurons and assessed the role of transcription factor PBX3 during neuronal lineage specification in the gut. Overexpression of PBX3 lead to an increased number of CALB1+ excitatory neurons and reduction of NOS1+ inhibitory motor neurons.

Thus, the work presented in this thesis shows that NEPTUNE enables rapid and flexible assessment of gene function during nervous system development.