Name: murine neural plate targeting by in utero nano-injection (neptune) at embryonic day 7.5
Uploaded: 2022-02-14T14:45:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5 at JoVE.com

We thank Bettina Semsch and Jia Sun (Infinigene) for expert care of mice; Florian Salomons and G&#246;ran M&#229;nsson from Biomedicum Imaging Core (BIC) for assistance with image acquisition and consultation. Funding: We thank the following funders for their support of this project: The Swedish Research Council, Karolinska Institutet (KI Foundations, Career Development Grant, Ph.D. student KID funding, and SFO StratNeuro funding, the Center of Innovative Medicine), The Ollie and Elof Ericssons Foundation, the Tornspiran Foundation, the Jeansssons Foundation, Sven and Ebba-Christina Hagbergs Prize and research Grant, Knut and Alice Wallenberg Project Grant, Fredrik and Ingrid Thurings Foundation, Lars Hiertas Minne, The Childhood Cancer Foundation (Barncancerfonden), The &#197;hlen Foundation, &#197;ke Wibergs Foundation, Tore Nilssons Foundation, and the Swedish Foundations Starting Grant to ERA. Figure 4D,E were created with BioRender.com.

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The authors declare no competing interests.

There are several steps in this protocol that influence embryonic survival, the quality of the injections, and the readout. The gestational age of embryos is defined as E0.5 at noon the day of the vaginal plug after overnight mating. Performing the ultrasound check for pregnancy at E6.5 in the late afternoon/evening ensures that the embryos are developed enough to be identified by ultrasound. The check (1) allows for prescreening of how many plug-positive mice are actually pregnant, (2) ensures no virus is thawed unnecessarily and wasted in the event of plug-positive mice not being pregnant, and (3) reduces unnecessary interventions on mice (avoids surgery on non-pregnant females).
At E7.5, embryos are sensitive to external forces and should be handled with care. For example, pulling on the uterine horns or squeezing the deciduas can lead to embryo resorption. The uterine tissue should always be kept moist when outside the female abdomen to prevent the tissue from drying. The majority of the deciduas should remain inside the female abdomen, with only 3-4 exposed for injections. Needle sharpness is another crucial determinant for successful injections. Blunt or broken needle tips result in repeated poking of deciduas or compression against the modeling clay before entering the AC, which can increase the resorption rate. Therefore, well-ground and sharp needles should always be stored safely and replaced after a maximum of 2 females.
This protocol describes how to target the neural plate with one single injection of lentivirus. Furthermore, it shows how transduction efficacy can be adapted from single-cell clones to the entire brain. However, other non-neural tissues, including the skin and oral epithelium, are targeted as well. In addition, all cell types (progenitors and differentiated cells) are transduced, making this approach efficient but nonspecific. The use of MiniPromoters in the viral construct leads to the specific expression of the transgene in neurons or astrocytes15. This has the advantage of avoiding the use of dedicated transgenic Cre animals and therefore reduces the amount of labor (strain maintenance and genotyping) and costs.
The limitations of NEPTUNE include its technical difficulty, challenges in obtaining pregnant females at a predictable and consistent rate, and the costs of acquiring specialized instrumentation. Furthermore, nonselective targeting of cells by lentivirus can be seen as both a benefit and a limitation of the technique. Injection of larger volumes into the AC results in exencephaly13, although brain malformations and exencephaly are avoided with the volumes described here15. A negative impact on brain development is thus a risk with in utero nano-injections that must be carefully avoided by injecting correct volumes adapted to the embryonic stage and AC size.
Future adaptations of the technique may focus on viral tropism. Adeno-associated viruses (AAVs) have different serotypes, which have been shown to robustly target different cell types in the CNS17,26. However, AAVs do not integrate into the host cell genome and therefore may be lost in cells with a high division rate. Although there are several ways to increase the specificity of NEPTUNE, transgenic animals are still the gold standard when it comes to gene manipulation in vivo. Cas9 mice and sgRNA-encoding lentivirus have been used for genetic screening in the embryonic epidermis27 and may also be adapted to the developing CNS.
Injections into the AC at E7.5 efficiently target cells of the neuroectoderm before initiation of neurulation and targets the developing brain more efficiently than in utero electroporation. This allows the study of genetic cues important for brain development from an earlier time point. In contrast to classical genetic mouse models, NEPTUNE offers a flexible approach to perform functional gene analysis. Phenotypes following overexpression or gene deletion can be studied within days to weeks compared to months or years. Injections of multiple viral constructs allow for the manipulation of several genes within one embryo and avoid the generation of double or triple knockout animals. Therefore, NEPTUNE not only saves time but also can reduce the number of animals used in research.

The brain and spinal cord are among the first organs to initiate formation during embryogenesis1,2. Although the genes associated with neurodevelopmental disorders are being identified, the functional interrogation of genetic variants has lagged behind3,4. As the generation of conditional knockout mice can take months or years, an alternative technique to rapidly investigate gene function in the developing brain is of interest. In mouse embryos, neurulation - the morphogenetic process by which the neural plate transforms into the neural tube to give rise to the central nervous system (CNS) - occurs between days 8 and 10 post conception5. Prior to the onset of neurulation, the neural plate, as part of the ectoderm, consists of a single layer of columnar cells that will proliferate and differentiate into the numerous neuronal and glial cell types within the CNS6,7. Therefore, to experimentally induce long-lasting alterations to gene expression in the CNS, targeting the neural plate offers obvious advantages, including the accessibility of all progenitor cells.
In neuroscience, in ovo electroporation8,9 and viral transduction of mouse embryos have been used to manipulate embryonic CNS gene expression. The developing chick embryo has been a model of choice for studying gene function during spinal cord development due to the accessibility of the chick embryo in the egg and the resultant ease of manipulating gene expression. In particular, in ovo plasmid electroporation generates experimental and control conditions in each chick spinal cord. Electroporation causes cell membrane permeabilization and directs negatively charged DNA away from the (negative) cathode towards the (positive) anode by applying an electric pulse via two electrodes to the embryo. In mice, in utero electroporation has generally been limited to embryonic stages at which neurulation has completed, and the brain or spinal cord already consists of several cell layers, resulting in low electroporation efficiency10. Plasmid electroporation results in transient gene expression and generally targets few cells.
Ultrasound-guided in utero microinjection has been used to manipulate different embryonic structures such as the skin and brain11,12,13,14. However, injections targeting the developing murine CNS have shown low efficacy or have negatively impacted embryonic survival12,13,14. Therefore, an improved protocol was developed for the delivery of high-titer lentivirus into the amniotic cavity (AC) at E7.5, which was dubbed NEPTUNE for neural plate targeting with in utero&#160;nano-injection15. Injections resulted in a long-lasting targeting efficacy of &#62;95% of the entire brain at E13.5. Furthermore, a staging step was introduced during ultrasound verification of pregnancy to sort females and pregnancies by developmental stage to minimize unnecessary procedures on research animals and maximize injection success. Injection efficiency and survival are tightly linked to the increase in AC size. Therefore, this paper describes how to measure AC size prior to injection to deliver a suitable volume to the AC that will not cause resorption of the embryo. NEPTUNE is a robust alternative to current in utero approaches and can be adapted for several uses, including, but not limited to, gain and loss of function studies, lineage tracing, or screening15,16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Bioscience</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G Needle</td>
			<td>BD Bioscience</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 inches capillaries</td>
			<td>Drummond Scientific</td>
			<td>3000203G/X</td>
			<td>Were used to pull in house needles</td>
		</tr>
		<tr>
			<td>70 MHz MS Series transducer</td>
			<td>Visual Sonics</td>
			<td>MS700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasonic clear ultrasound gel</td>
			<td>Parker Laboratories</td>
			<td>Mar-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip Applier 9 mm</td>
			<td>Angthos</td>
			<td>12020-09</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 mice</td>
			<td>Charles River, Germany</td>
			<td>Crl:CD1(ICR)</td>
			<td>Females: from age of 8 weeks old 
			Males: from the age of 12 weeks old</td>
		</tr>
		<tr>
			<td>Cotton Swab</td>
			<td>OneMed Sverige AB</td>
			<td>120788</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps delicate straight 13 cm</td>
			<td>Agnthos</td>
			<td>08-032-130</td>
			<td></td>
		</tr>
		<tr>
			<td>EG-400 Narishige Micropipette Grinder</td>
			<td>Narishige</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ clips 9 mm</td>
			<td>Angthos</td>
			<td>59027</td>
			<td>clips</td>
		</tr>
		<tr>
			<td>Iris Scissors, Super Cut, straight, 9 cm</td>
			<td>Agnthos</td>
			<td>307-336-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Baxter Medical AB</td>
			<td>EAN: 50085412586613</td>
			<td>Purchased from Swedish Pharmacy</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly Clarke</td>
			<td>7557</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Tape</td>
			<td>Visual Sonics</td>
			<td>SA-11053</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Modeling Clay</td>
			<td>Sense AB</td>
			<td>10209</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Handling Table</td>
			<td>Visual Sonics</td>
			<td>50249</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto Injector Kit</td>
			<td>Drummond</td>
			<td>3-000-205A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>HS234526C</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish with central opening (low wall)</td>
			<td>Visual Sonics</td>
			<td>SA-11620</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, (&#216;xH): 92 x 16 mm</td>
			<td>Sarstedt</td>
			<td>82.1472.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rely+On Virkon</td>
			<td>DuPont</td>
			<td>130000132037</td>
			<td>disinfectant</td>
		</tr>
		<tr>
			<td>Silicone membrane</td>
			<td>Visual Sonics</td>
			<td>SA-11054</td>
			<td></td>
		</tr>
		<tr>
			<td>Steri 250, hot bead sterilizer</td>
			<td>Angthos</td>
			<td>31100</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tape (1.25 cm x 9.14 m)</td>
			<td>Medicarrier</td>
			<td>67034</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Compact Dual (Med. Air &#38; O2) Anesthesia System</td>
			<td>Visual Sonics</td>
			<td>VS-12055</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Imaging Station 2</td>
			<td>Visual Sonics</td>
			<td>VS-11983</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo2100</td>
			<td>Visual Sonics</td>
			<td>VS-20047</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 6-0; C-3 needle, 45 cm purple filament</td>
			<td>Agnthos</td>
			<td>J384H</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine neural plate targeting by in utero nano-injection (neptune) at embryonic day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Embryos injected at E7.5 with hPGK-H2B-GFP lentivirus11,12 were collected at E13.5 and examined under the fluorescent dissection microscope (Figure 5A). Successful transduction of the neural plate results in embryos with strong expression of a fluorescent reporter in the brain mainly and in other ectoderm-derived tissues, e.g., the skin (Figure 5A,B). Injection of an excessively high volume (greater than the volumes recommended here, e.g., &#8805;500 nL) increases the pressure in the AC and can result in either complete resorption (data not shown) or neural tube defects such as exencephaly (Figure 5A). Successful injections at E7.5 result in uniform transduction from the forebrain to the hindbrain (Figure 5C-J).
Lentiviral titers around 2 &#215; 1010 infectious units (IFU) achieve over 95% targeting, while titers of ~1 &#215; 109 IFU achieve 15% targeting efficiency15. In addition, structures that have previously been difficult to target using electroporation, such as the choroid plexus17,18, are also targeted (Figure 5 E,F). Transduction efficacy can be modified by adjusting the viral titer delivered into the AC. Injections of low titer result in the transduction of single-cell clones (Figure 5C, Figure 5E, and Figure 5G,H) while usage of high-titer virus transduces nearly 100% of the entire brain (Figure 5D, Figure 5F, Figure 5H, and Figure 5J). Therefore, NEPTUNE can be used for either clonal transduction, lineage tracing, and genetic screening approaches or studying the global effects of gene overexpression or downregulation in the entire brain.
Mammalian eye development is the result of well-organized communication between three derivatives of the embryonic ectoderm: the neural retina (NR) and retinal pigment epithelium (RPE) are derived from the neuroepithelium of the ventral forebrain, while the surface ectoderm gives rise to the future lens and corneal epithelium. However, the central stroma and the posterior endothelium, the other two layers of the cornea, are derived from neural crest cells of the periocular mesenchyme19,20. Coronal sections through E13.5 embryos, injected at E7.5 with high-titer lentivirus showed, similar to the brain, high and uniform transduction of the neural tissue of the eye, as well as the lens, cornea, and mesenchyme (Figure 5K). As neurulation progresses, injections at E8.5 and E9.5 result in the continued targeting of the lens and corneal epithelium (Figure 5L and Figure 5N), while transduction of the neuroectoderm-derived tissues of the eye is less efficient at E8.5 (Figure 5L,M) or not targeted at E9.5 (Figure 5N).
While most viral particles infect the exposed tissues upon injection, some particles transduce non-ectodermal derived tissues that develop later (Figure 6A). Salivary glands and ducts develop around E11.5 from the oral epithelium21 and are targeted with NEPTUNE (Figure 6B). Following injections at E9.5, the lingual epithelium of the tongue is well transduced; however, the underlying mesenchyme is negative (Figure 6C; brackets denote transduced cells; asterisks denote autofluorescence signal not transduced cells). In addition, there are positive clusters within the lingual epithelium, separated by negative sections (Figure 6C, white arrowheads), suggesting transduction of the papilla surface. Neural crest cells have been described in the underlying tongue mesenchyme and within the lingual epithelium, where they are involved in the development of taste papillae and taste buds22. Indeed, injections at E7.5 result in widespread transduction of the tongue mesenchyme at E13.5 (Figure 6D), suggesting that early injections target neural crest cells, contributing to mesenchyme in the tongue.
In vertebrates, the dorsal root ganglia (DRGs) are a central component of the peripheral nervous system (PNS), as all somatosensory input from the body&#39;s periphery (temperature, pain, pressure) is transmitted to the brain via activation of the DRG neurons23. Both neurons and glial cells of the DRG are derived from trunk neural crest cells24. Lentiviral injection, in which the ubiquitous hPGK promoter controls expression of the fluorescent reporter, leads to widespread targeting of the central and peripheral nervous system (Figure 7A), transducing both neurons and progenitors in the spinal cord (Figure 7B), as well as the DRG (Figure 7C). Using a MiniPromoter for doublecortin25 makes it possible to limit GFP expression to neurons only (15 and Figure 7D,E).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63148/63148fig5v3.jpg" /> 
Figure 5: High efficiency or clonal transduction with NEPTUNE. (A) E13.5 embryos under a dissection microscope illuminated with standard lighting (left panel) and same embryos illuminated for GFP (right panel). Uninjected embryo on the far left, successfully injected embryo on the far right, yielding positive signal in the brain (rightmost embryo). Exencephalic embryo due to excess volume injected (middle embryo). (B) Skin and brain targeting with E7.5 injection. Scale bar = 50 &#181;m. (C, D) E13.5 confocal images of forebrain with low clonal transduction (C) or high-efficiency transduction (D). (C, E, G, I) Clonal transduction of different regions in brain. (D, F, H, J) High-efficiency transduction of different regions in brain. Scale bars = 200 &#181;m. Different transduction efficacies are representative of other areas of the CNS. (E, F) Choroid Plexus targeting, clonally (E) or with high efficiency (F). Scale bars = 200 &#181;m. (G, H) Clonal targeting (G) and high-efficiency (H) targeting of hindbrain, magnified in (I, J). Scale bars = 200 &#181;m. (K) GFP reporter expression in eye at E13.5 after in utero injection at E7.5 (L, M) GFP reporter expression in eye at E15.5 after in utero injection at E8.5 (L), boxed region magnified in (M), or at E9.5 (N). Autofluorescent blood vessels are marked with white stars. Scale bars = 100 &#181;m. Abbreviations: NEPTUNE = neural plate targeting with in utero&#160;nano-injection; C = Cornea; LE = Lens Epithelium; LF = Lens fibers; M = Mesenchyme; NR = Neural Retina; ON = Optic Nerve; RPE = Retinal Pigment Epithelium; GFP = green fluorescent protein; CNS = central nervous system. <a href="https://www.jove.com/files/ftp_upload/63148/63148fig5v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63148/63148fig06.jpg" /> 
Figure 6: In utero transduction of non-neural tissues. (A) Confocal image of E15.5 oral cavity. Embryo was injected with fluorescent reporter lentivirus at E9.5. Scale bar = 200 &#181;m. (B, C) (B) Magnification of inset panel; salivary duct epithelium transduced with virus. (C) Magnification of inset panel; dorsal lingual epithelium transduced with GFP reporter virus (white bracket). The underlying mesenchyme derived from neural crest cells is negative. White arrowheads indicate papillae with negative neural crest cells surrounded by virus-transduced epithelial cells (autofluorescent blood cells/vessels are marked with white stars). Scale bars = 50 &#181;m. (D) Schematic and confocal image of E13.5 tongue mesenchyme after injections with fluorescent reporter virus at E7.5. Scale bar = 50 &#181;m. Abbreviations: D = Dorsal; M = Mesenchyme; N = Nasopharynx; SLD = Sublingual Duct; SMD = Submandibular Duct; T = Tongue; V = Ventral; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/63148/63148fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63148/63148fig07.jpg" /> 
Figure 7: Transduction of neural crest-derived dorsal root ganglion cells. (A) NEPTUNE targeting at E7.5 allows targeting of both CNS and PNS. (B) Confocal image of E13.5 spinal cord and DRGs injected with hPGK-H2B-GFP reporter lentivirus at E7.5 shows transduction of both SOX2+ neural progenitors and NeuN+ neurons. Scale bar = 100 &#181;m. (C) Boxed region of B showing the DRG with GFP expression in SOX2+ (white arrows) and NeuN+ (white arrowheads) cell populations. Scale bar = 20 &#181;m. (D) Confocal image of E13.5 spinal cord and DRG injected with DCX-H2B-GFP lentivirus at E7.5, targeting only DCX+ cells. Scale bar = 100 &#181;m. (E) Boxed region of D showing DRG with GFP expression restricted to DCX+ neurons (white arrowheads). DCX- cells are negative for GFP (white arrows). Scale bar = 20 &#181;m. Abbreviations: NEPTUNE = neural plate targeting with in utero&#160;nano-injection; CNS = central nervous system; PNS = peripheral nervous system; DRGs = dorsal root ganglia; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/63148/63148fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5 at JoVE.com

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

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