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Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure

Katrin Mangold, Jan Mašek, Jingyan He, Urban Lendahl, Elaine Fuchs, Emma R Andersson

Preprint posted on May 28, 2020 https://www.biorxiv.org/content/10.1101/2020.05.28.115972v1

Men are from Mars, but transgenic mice are from NEPTUNE. NEural Plate Targeting by in Utero NanoinjEction as a new method to genetically modify the central nervous system using lentivirus.

Selected by Martin Estermann

Background:

Nowadays, thousands of genetically engineered mouse mutants are available to study the function of specific genes or to model diseases. However, the process of generating mouse mutants is time consuming and involves several breeding steps. Due to this problem, the research community started to use other non-mammalian models with faster lifespan and external development, like fish and birds, to address evolutionary conserved mechanisms in embryogenesis. The development of new technologies, like CRISPR, accelerated the process of transgenic mice generation, but it still relies on generating adult mice strains for breeding.

Genetic manipulation of the developing mouse brain has mostly relied on in utero electroporation, especially after neurulation, which results in targeting only a small subset of brain cells. In utero amniotic fluid transduction with lentiviruses demonstrated a non-invasive and highly efficient transduction of mouse embryonic surface epithelium. This technology relies on the infection of cells that are in contact with the amniotic fluid.

The neural plate, which gives rise to the central nervous system (CNS), is in contact with the amniotic fluid in the uterus making it an ideal tissue to target by in utero lentiviral injection. In this preprint the authors developed NEPTUNE: NEural Plate Targeting by in Utero NanoinjEction to modify gene expression in the neural plate, prior to neurulation and achieve a stable transduction of the mouse embryonic CNS.

 

Key findings:

1) NEPTUNE allows stable transduction of the murine central nervous system

The amniotic cavity was injected at embryonic day (E) 7.5, prior to neurulation, with lentivirus, allowing the neural plate cells enough exposure to virus to get infected (Fig. 1A). Embryos were then collected at E13 to assess the transduction efficiency by immunofluorescence and flow cytometry (Fig. 1B). These experiments showed that more than 95% of the cells in the developing central nervous system (CNS) could be targeted by NEPTUNE. This transduction was even across the anterior to posterior regions of the brain and also across the different brain cell types, including the cerebellum. In addition to the CNS, other organs were targeted, such as skin, lung, gastrointestinal tract, liver and heart, mainly due to their exposition to the amniotic fluid or because they are derived from the neural crest (Fig. 1B).

In addition, to test if the transduction was stable, 6 months old mice brains were collected and assessed for fluorescence, confirming that the transduction was stable at least 6 months after the in utero viral injection (Fig. 1C).

Fig. 1. NEPTUNE results in widespread and stable transduction of the murine CNS. (A) Lentivirus containing a GFP expressing vector was injected in the amniotic cavity at E7.5. (B) Embryos were collected at E13 and assessed for GFP expression. (C) 6 months old mice brains were collected and assessed for GFP expression. (Preprint Fig. 2A-C).

2) Using mini-promoters to regulate cell type specific expression by NEPTUNE

NEPTUNE was able to target the entire central nervous system and other organs as well. In order to achieve a precise targeting of specific cell types, the authors used previously identified mini-promoters to drive GFP expression in different neural cells. They used the Dcx mini-promoter to target immature neurons, Olig1promoter to target oligodendrocytes and Gfap promoter to target astrocytes (Fig. 2A). Brain specific GFP expression was detected in the Dcx embryos (Fig. 2A Right), compared with the widespread expression of a constitutive promoter vector (Fig. 2A Left).

In addition, to validate the cell specificity of the constructs, immunofluorescence for DCX, OLIG1 and GFAP was performed to show co-expression of GFP with the different cell type specific markers. The neural stem cell marker SOX2 was used as a negative immunofluorescence control. As expected, the promoters were able to only drive GFP in their specific cell types, only colocalizing with their respective cell type marker (Fig. 2C). In addition, none of the evaluated promoters showed GFP expression in SOX2+ neural progenitors (Fig. 2C), demonstrating that NEPTUNE gene expression can be directed using cell specific type mini-promoters.

Fig. 2. Cell specific expression of GFP was achieved combining mini-promoters and NEPTUNE. (A) Lentivirus constructs generated where global promoters (left) or cell specific cell mini-promoters (right) were used to drive GFP expression. Lentivirus were injected in the amnion cavity and embryos were collected and assessed for GFP expression. (B) Immunofluorescence against DCX (immature neurons marker), OLIG1 (Oligodendrocytes marker) and GFAP (astrocytes marker) and GFP was performed to evaluate cell specific expression of GFP. SOX2 (neural progenitor marker) was used as a negative control. DAPI was used as nuclear staining. (Preprint Fig. 5A and C).

3) NEPTUNE can be used to efficiently knock down genes by shRNA

The authors have shown that gene overexpression was possible, either globally, in the CNS, or in a cell-type specific manner. To address if gene dysregulation was also possible using NEPTUNE they used shRNA to knock down Sptbn2, a gene highly expressed in the CNS, for which a loss of function phenotype was never achieved or studied. Current mouse models for Sptbn2 express shorter isoforms or alternative splice variants, resulting in defects in motor functions, suggesting that the phenotype might not reflect a loss of function. NEPTUNE shRNA injection resulted in an increased embryo lethality, with none of them surviving E18.5 (Fig. 3D). Embryos collected at E9.5 (Fig. 3E) and E13.5 (Fig. 3F) showed a more severe phenotype indicating that Sptbn2 is crucial for neural tube development and abdominal wall closure, and that the reduction of Sptbn2 expression is embryonic lethal. In addition, these results demonstrate the strong capability of the NEPTUNE injection system to silence CNS gene expression during embryonic stages.

Fig. 3 Sptbn2 knockdown in vivo with NEPTUNE reveals a novel role in neurulation and abdominal wall closure. (D) Survival rates Sptbn2 knockdown mouse embryos collected at E9.5, E13.5 and E18.5. (E) E9.5 shRNA injected embryos showed neurulation/turning defects (white arrowheads). (F) E13.5 Sptbn2 knockdown embryos displayed halted turning (white arrowheads) and abdominal wall defects (black arrowheads). (Preprint Fig.6 D-F).

Why I choose this paper:

Mouse is the most used mammalian research organism, but the generation of transgenic mice is a time-consuming task that involves transgenerational breeding. Due to this reason, other non-mammalian models with external development, like fish and birds, are frequently used to address embryonic evolutionary conserved mechanisms. This preprint introduces a new methodology to target the CNS by lentiviral in utero injection, allowing to test gene function rapidly and efficiently during embryonic development, without the necessity of breeding. This will not only reduce the time of genetic screening but also would be crucial to study the phenotype of embryonic lethal genes. This methodology also showed that genetic expression could be regulated in a cell specific manner and has the flexibility to couple with other mouse genetic tools. In addition, not only the CNS was targeted, but also other organs in contact with the amniotic fluid, like lungs and gastrointestinal system, allowing this system to be expanded to other research areas.

 

Future directions / questions for the authors:

  • Most of the features that were included in the vectors were relatively small, for example the utilization of shRNA, mini-promoters and the suggestion of using lentivirus as guide delivery in CAS9 expressing mice. Is there any size limitation when using this lentiviral system? Can the system be used to overexpress proteins others than GFP? What is the largest protein size that could be overexpressed?
  • Were you able to identify any CNS cell type or region that was not targeted or was harder to target compared to others?
  • Is it possible to inject two different viral constructs by NEPTUNE? Do you expect them to compete with each other or will they target the same cells simultaneously? In addition, how feasible would it be to combine NEPTUNE with the brainbow system in a CRE expressing mouse?

 

Posted on: 24th June 2020

doi: https://doi.org/10.1242/prelights.22142

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