Essay: Neural tube defects

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Introduction
Neural tube defects (NTDs) are severe birth defects of the central nervous system and refers to failed neural tube closure during embryonic development (Ghi et al., 2018). The phenotype presented by those with NTDs is dependent on where the neural tube remains open (Copp and Greene, 2010). Therefore, the seriousness of disease manifestation varies. There are specific signalling pathways which are essential for neural tube closure: non-canonical Wnt/planar planar cell polarity (PCP) pathway, sonic hedgehog (SHH) pathway, bone morphogenic protein (BMP) signalling, notch signalling, retinoid signalling and inositol metabolism (Nikolopoulou et al., 2017).

Initiation of neural tube formation studied in model organisms involves a convergent extension (CE) process whereby a tissue narrows along its mediolateral axis and elongates along its anteroposterior/rostrocaudal axis (Nikolopoulou et al., 2017; Chen et al., 2018). This allows modelling of the neural plate before closure of the tube. The PCP pathway is responsible for CE and mutants in which PCP signalling is disturbed have shown NTDs caused by aberrant CE. This caused the widening of the neural plate, so the neural folds could not fuse at the midline. Greene et al., 1998 first described failed neural tube closure in mice carrying mutations in genes such as VANGL2 essential in the PCP pathway. Homozygosity of this gene causes craniorachischisis whereby the neural tube remains open throughout the midbrain, hindbrain and entire neural tube (Murdoch et al., 2001). Therefore, PCP signalling pathway is critical for establishing and maintaining planar polarity during morphogenesis.

SHH is a morphogen of the SHH signalling pathway essential for the formation and patterning of the midline structures in the brain, spinal cord and forebrain (Garcia et al., 2018). Apart from neuronal structures, SHH is involved in cell growth, cell specialisation, normal patterning of the body and eye formation. Cells that develop into the eyes originate from the centre of the developing face at a single structure called the eye field. SHH causes the eye field to separate into two distinct eyes (Zagozewski et al., 2014). Holoprosencephaly, spina bifida and exencephaly are NTDs associated with aberrant SHH signalling (Murdoch and Copp, 2010). Spina bifida manifests when increased SHH signalling blocks the formation of dorsolateral hinge points. This would not occur when reduced SHH signalling ensures the dorsolateral hinge points to properly close along the entire body axis (McShane et al., 2015).

In the absence of SHH, Patched1 (Ptc1) inhibits the membrane protein Smoothened (Smo). Binding of SHH to Ptc1 removes the inhibitory effects of Smo to initiate an intracellular signalling pathway. Intraflagellar transport is an important component of the vertebrate SHH pathway to facilitate the movement of proteins from the cell into the primary cilium. When the SHH pathway is activated, Smo is localised on the distal top of the primary cilia. Ptc1 inhibits the endocytic internalisation of Smo into the cilium. Gli transcriptional activators enter the nucleus upon Smo activation to control the transcription of SHH target genes (Murdoch and Copp, 2010). However, NTDs seen in mice as a result of faulty SHH signalling is usually due to deleterious genetic changes in proteins that influence the SHH pathway (Copp and Greene, 2010). For example, NTDs in mice can manifest mutations in Ptc1 and when other SHH inhibitory genes such as Gli3 and Tulp3 lose function (Patterson et al., 2009). Although NTDs are not produced from loss of function in proteins such as Smo and SHH which activate signalling (Gigante et al., 2018), neural tube closure might be compromised when such proteins are over-expressed. This results in failure of the neural tube to bend and therefore causes NTDs in the midbrain and lower spinal regions.

Additionally, the notch signalling pathway is involved in proliferative signalling during neurogenesis in most multicellular organisms. Via lateral induction and lateral inhibition respectively, notch signalling establishes the neural crest domain and differentiates cells originating in the neural crest (Cornell and Eisen, 2005). Furthermore, Main et al., 2013 reported the effects of homozygous mouse mutants deficient in notch signalling. Cell polarity and CE defects attributed to failed apical/basal polarity in the neural tube were exhibited in the phenotypes of the mutant mice. Main et al., 2013 also found pharmacologically inhibited wild-type mice showed accelerated neuronal differentiation and neural rosette breakdown. Neural rosettes are radially organised columnar epithelial cells that form during neural differentiation (Harding et al., 2014) and are maintained by the notch and SHH pathways (Elkabetz et al., 2008). However, disrupting the actin filaments and Rho kinase (ROCK) activity needed for the structural integrity of neural rosettes did not affect on neuronal differentiation. This showed that maintaining rosette integrity is not a requirement for normal neuronal differentiation.

Rho guanine nucleotide exchange factor (Rgnef) is a ubiquitous 190kDa RNA binding protein containing a domain for RhoA activation. Although it is unknown the exact role of Rgnef, it is thought that the small GTP-binding protein Rho is involved in controlling neuronal morphology (Gebbink et al., 1997) and specifically involved in controlling signalling pathways regulating cell proliferation and movement (Miller et al., 2014). Rgnef is involved in the formation of neuronal cytoplasmic inclusions in degenerating spinal motor neurons, indicative of amyotrophic lateral sclerosis (ALS) (Strong et al, 2005). Rgnef is unique in being a signal transduction protein and RNA-binding protein. This means it is able to transduce signals through guanine exchange activation of RhoA and affect the stability of low molecular weight neurofilament mRNAs. Rgnef mutations have been found to contribute to the manifestation of ALS through aggregation with RNA-binding proteins and fusing within sarcomas within spinal motor neuron neuronal cytoplasmic inclusions in ALS (Zhang et al., 2016). Furthermore, previous experiments in the Murdoch lab (unpublished) identified Rgnef and the Tubby-like-protein 3 gene (Tulp3) were found to interact in a yeast 2-hybrid screen. This showed Rgnef and Tulp3 at least had the capacity to interact with each other. When Tulp3 malfunctions, the SHH pathway is activated downstream and ventral signalling increases, affecting patterning. This led to initial propositions that the Rgnef was a mediator for proper Tulp3 functioning. This link between Tulp3 and Rgnef, along with the activation of SHH pathway and the NTDs caused from aberrant SHH signalling provided the basis of this reports’ investigation

This study used in situ hybridisation (ISH) in the localisation of Rgnef gene within a histological section of mouse embryos at embryonic days (E) 10.25 to 16.5 days. This was achieved using riboprobes complementary to the target mRNA species. Non-radioactive ISH using digoxigenin (DIG)-labelled RNA probes are often used because they localise selected mRNA species and are easy to produce by PCR (Schaeren-Wiemers and Gerfin-Moser, 1993). The DIG-labelled probe is detected using an alkaline phosphatase conjugated anti-DIG antibody and immunodetected using a chromogenic substance such as nitro-blue tetrazolium and 5-bromo-4-chloro-3’-indolyphosphate (NBT/BCIP) against low background staining (Karcher, 1995).

Riboprobes produce RNA-RNA hybrids when bound to mRNA in the sections. This is advantageous because they are extremely thermostable and resistant to digestion by RNases. This allows the washing of non-hybridised RNA from the section to reduce background staining (Darby, 2004). Riboprobes are extremely sensitive to RNases which degrade RNA, so it is important to use sterile techniques when preparing them. In this experiment, the riboprobes were synthesised using in vitro transcription using linearised plasmid DNA. Riboprobes can be generated by Sp6 and T7 RNA polymerases associated with transcription initiation and elongation factors in the sense and antisense directions (Meyer and Grainger, 2013).

This report addresses the expression of Rgnef in mouse embryonic development from E10.25 to E16.5. This was achieved through assessing staining on different embryonic sections through in situ hybridisation and comparing the expression data of Rgnef to SHH.

Materials and Methods
All materials were obtained from either Sigma or Fisher unless stated otherwise.

Primer Design for PCR
Eurofins Genomics’ PCR Primer uses Prime+ software to design the primers involved in the experiment. Oligonucleotide primers were selected from the Rgnef DNA and used as a template sequence. Upper and lower limits for primer and product Tm and GC content. The software ensured that primers with extensive self-complementarity were avoided to minimise primer secondary structure and primer dimer formation. Design parameters took the complete sequence as the target region and C/G as the primer 3’ clamp, while the maximum Tm difference between the primers was set at 2C. The software produced three sets of forward and reverse primers. Each set contained a different GC content and different Tm. The results of the primer design, as well as the primer design of a colleague can be seen in Table 1.

Making Primer Solution from Dry Primers
Forward and reverse primer solutions were made up from dry primers to 200 M using Tris-EDTA (TE) buffer. PCR was carried out using a temperature gradient from 55.0 – 62.0C in order to establish the optimum annealing temperature of the primers to the target DNA region. Six reactions were carried out using 5X Green GoTaq Flexi Buffer, 25 mM Mg2+, 10 mM dNTPs, forward primer, reverse primer, Taq polymerase, cDNA and ddH2O. These reactions were incubated at 95C for 3 minutes, then underwent 31 cycles at 95C for one minute, temperature gradient for 45 seconds, followed by another incubation at 72C for 40 seconds and then one final incubation at 72C for 10 minutes.

Gel Electrophoresis of PCR Samples
In order to test whether the target DNA sequence had been annealed, the DNA from the six reactions were run on a 1% agarose gel in 1X TAE (Tris base, acetic acid and EDTA) buffer at 120V for 30 minutes.

Purification of PCR Samples for Ligation
PCR products were purified for ligation using Qiagen’s QIAquick PCR Purification Kit. 900 l Buffer PB (5 M Gu-HCl, 30% isopropanol) was added to 180 l PCR sample and placed in a QIAquick spin column within a collection tube. This was centrifuged for 1 minute to bind DNA. The flow through was discarded and 750 l the DNA was washed with Buffer PE (10 mM Tris-HCl, pH 7.5, 80% ethanol) and the column was centrifuged. The flow through was discarded and the column was centrifuged for 1 minute. 50 l Buffer EB (10 mM Tris-HCl, pH 8.5) was added and centrifuged for 1 minute to elute DNA. Water and loading dye were added to 3 l of the product and run on the gel for 80V for 20 minutes.

Ligations were set up using a 2X rapid ligation buffer, T4 DNA ligase, pGEM-T easy vector (50 ng), PCR product, T4 DNA ligase (3 Weiss units/l), ddH2O. This was in a 1:3 (vector : insert) ratio. The reaction was incubated for 1 hour at room temperature.

Pouring LB Agar Plates and Transformation of E. coli
The selective antibiotic ampicillin was added to the LB agar. Sterile conditions were used to pour the LB agar into the plates. Cells were taken and 1l Shh DNA, 1l water and 5l Rgnef DNA were added to different cells. The cells were incubated for 30 minutes on ice. The cells were then heat shocked in a 42C for 40 seconds. 0.5 ml LB broth was added to the transformation reaction. The cells were placed in a 37C shaking incubator at at 200 rpm for 45 minutes. The transformed cells were plated on the LB agar plates containing the selective antibiotic. The plates were then incubated overnight at 37C. Individual colonies were chosen from each transformation for incubation with ampicillin in LB broth at 37C on the shaking platform for 2 hours. PCR then confirmed whether the DNA was transformed.

Midi-prep of Samples and DNA Elution
For unforeseen circumstances, my lab partner carried out this section of the protocol.

DIG-Labelled Probe Synthesis
RNAse free conditions were needed for the DIG-labelled probe synthesis step. A plasmid for probe synthesis was generated using the Qiagen Midiprep procedure and 10 g of plasmid was linearised with the appropriate restriction enzyme. The linearised DNA was then precipitated by adding 1/20 vol 3M sodium acetate and 2 vol ethanol and left in the freezer at –20C for 1 hour. This was spun down in a centrifuge at 13,000 rpm for 10 minutes at 4C. The pellet was washed with 70% ethanol and spun again to remove the supernatant. After air drying, the pellet was resuspended in RNAse free water at 0.5 g/l. A probe labelling reaction using Fermentas enzymes was set up using the following constituents: 5 l linearised plasmid (0.5 g) in 9.5 l RNAse free water, 2 l 10X transcription buffer, 2 l DIG labelling mix, 0.5 l RNAse inhibitor (100 U/l) and 1 l Sp6 and T7 RNA polymerase (10 U/l). The reaction was then incubated at 37C for 2 hours. The probe was precipitated by adding 100 l RNAse free TE, 10 l 4M lithium chloride and 300 l ethanol. This was then incubated at –20C for 2 hours. A 1 l aliquot of the probe reaction was run on 1% agarose gel to estimate the amount of probe synthesised. The probe was then spun down in the centrifuge at 13,000 rpm for 20 minutes at 4C. The pellet was immediately resuspended in 100 l RNA free TE to make the probe around 0.1 g/l and then stored at –20C.

DIG in situ from Wax Sections
Three days before starting the in situs, all appropriate solutions were treated with 0.1% diethyl pyrocarbonate (DEPC) overnight to inactivate RNAses and autoclaved at 121C for 15 minutes to break down the DEPC.

Pre-hybridisation treatments
The embryo sections were dewaxed and rehydrated using histoclear, twice, for 10 minutes each and rehydrated using an ethanol series decreasing from 100% to 50% for 2 minutes per wash. The sections were fixed using 4% PFA in PBS for 20 minutes and then washed twice in PBS for 2 minutes, twice. Next, 20 g/ml proteinase K in PBS was used to hydrolyse peptide bonds within the cell to allow insertion of the RNA probe. Then, a second fixing step occurred using 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 5 minutes and washed in PBS twice for 2 minutes. Next, 1 ml acetic anhydride was used for the acetylation step and stirred for 10 minutes, followed by a wash in PBS, twice for 2 minutes. The sections underwent a dehydration step using an ethanol series increasing from 50% to 100%. HybMix (50% deionised formamide, 0.3 M sodium chloride, 20 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8.0, 10% dextran sulfate and 1X Denhardts solution) was added to 1g/ml RNAse inhibitor, 0.5 mg/ml yeast tRNA and specific probe at a 1:100 dilution. This was denatured at 70C and quenched on ice. Slides were laid out on aluminium foil and the HybMix was added. The slides were placed in the oven at a hybridisation temperature (65C) and kept moist using 50% formamide with 2X saline-sodium citrate (SSC).

Post-hybridisation washes
Slides were washed with 2X SSC for 15 minutes at 65C and twice with formamide wash at 65C for 20 minutes. Slides were washed twice with 2X SSC for 30 minutes at 65C and twice with 0.2X SSC for 30 minutes at 65C. After leaving to cool at room temperature the slides were washed with buffer B1 (0.1 M Tris-HCl pH 7.6, 0.15 M sodium chloride), twice for 5 minutes each. Non-specific binding was blocked by applying filtered 10% Newborn Calf Serum (NCS) in buffer B1 and left at room temperature for 1 hour in humidified chamber. Anti-DIG antibody diluted 1:1000 in 2% NCS in buffer B1 was added to the slides. A parafilm coverslip was applied and slides were left in humidified chamber overnight at 4C.

Antibody Detection and Colour Development
Slides were washed with buffer B1 three times for 3 minutes each and buffer B2 (0.1 M Tris-HCl pH 9.5, 0.1 M sodium chloride) twice for 10 minutes each. NBT/BCIP in buffer B2 was added to each slide in a light-proof humidified chamber. This was left at room temperature for 4 hours at which point a colour development was observed and left at 4C overnight. After complete development, the slides were placed under a running tap for 10 minutes and dehydrated quickly with 50% – 100% ethanol series for 8 seconds and placed in histoclear twice for 5 minutes. The slides were mounted with coverslips and viewed under a microscope.

Results
In this experiment, histological segments of mouse embryos were analysed for Rgnef expression in ages ranging from 10.25 to 16.5 days post conception. In situ hybridisation is a technique that allows for the precise localisation of specific nucleic acid segments within a histological section. Nucleic acids are suitable for detection when a complementary riboprobe is bound with a reporter molecule attached. This allows assessment of the degree of Rgnef expression within the mouse embryo and visualisation of the specific sites and organs in which Rgnef is expressed (Brown, 1998).

Primer Design
First, primers were designed that were complementary to regions flanking only Rgnef DNA fragments. The Mus musculus Rgnef gene is 6259 bp long and contains a coding sequence from positions 105 to 5207 (Figure 25). The coding sequence is a section of DNA that leads to production of translated RNA (Brenner et al., 2002).

This experiment used a novel probe with the aim to generate more robust expression data. There were several factors for consideration when designing primers including a balance between specificity and amplification efficiency. Specificity is the frequency a mispriming event might occur. Poor specificity results in unfavourable amplification of the product, so will not produce the correct DNA clone. Amplification efficiency refers to how close a primer pair amplifies a product to its theoretical optimum of a twofold increase of product for each PCR cycle (Dieffenbach et al., 1993). Eurofins Genomics’ PCR Primer Design Tool automatically controlled the balance between these two variables. The length of a primer has a direct relation to its specificity. All the forward and reverse primers designed for this experiment had a length of twenty bases, 5’ end stability, 3’ end specificity, GC content between 50 – 60% and Tm between 60 – 64C (Table 1), in which Tm of primers was calculated approximately as Tm = 4 (G + C) + 2 (A + T). These parameters allowed for a primer design which would theoretically be successful. The GC content and Tm of primers are other important factors in primer design in order to provide an appropriate thermal window for efficient annealing (Dieffenbach et al., 1993). At Tm too low, there is a loss of specificity. At a Tm too high, there is an increased chance of mispriming. Finally, secondary structures of primers needed to be assessed when designing them (Singh et al., 2000). Primers need to be non-complementary to each other, especially at the 3’ end. Self-complementarity can lead to the formation of hairpin loops or primer-dimers (Singh et al., 2000). A hairpin loop is created when unpaired regions in a sequence forms base pairs with another section of the same strand (Hairpin Loop (mRNA), 2014). Hairpin loops limit the availability of its nucleotides to bind to the complementary nucleotides on the Rgnef DNA strand. Primer-dimers are a PCR by-product consisting of primer molecules that have hybridised together due to complementary bases between the forward and reverse primers (Dieffenbach et al., 1993).

Synthesis of Rgnef gene using Polymerase Chain Reaction
Furthermore, PCR was used to synthesise multiple copies of the Rgnef gene (Figure 28). However, there were some initial problems with the PCR process. There were no bands in any of the lanes and thus no PCR product present when using cDNA (Figure 17). Therefore, the PCR was repeated using genomic DNA to observe whether the type of DNA used was the issue in the PCR process (Figure 18) and similar results were observed. Again, there was no PCR product on the gel. This prompted further adaption of the PCR protocol in order to determine the cause of the negative results. It was determined that the dNTPs initially used might have degraded due to their age or storage conditions. Therefore, the old dNTPs were amplified along with a new set of dNTPs using a pBluescript plasmid and ran on an agarose gel. Four bands were observed, all of expected size for the four old dNTPs (dATP, dGTP, dTTP and dCTP) (Figure 19a). There were also four bands of expected size for the new dNTPs (Figure 19b). This shows that the dNTPs initially used were not the problem in the PCR process. Therefore, another PCR was carried out in which different types of DNA were amplified to determine whether the DNA samples previously used had degraded.

There are definitive bands for some of the DNA samples (Figure 20). At 55C, only crc/CBA and hhkr1 are visible for the first primer set. At 60C, A3, 197.2a, crc/CBA and hhkr1 are visible for the first primer set. At 55C, A3, 197.2a, crc/CBA and hhkr1 are visible for the second primer set. At 60C, A3, 197.2a, crc/CBA and hhkr1 are visible for the second primer set. Only crsh/crsh did not present any visible bands at either temperature for either of the first or second primer sets. On the other hand, there were no visible bands for the third primer set. Only one clear band can be seen for the fourth primer set for 197.2a4 DNA at 55C. There is one faint band at 55C for primer four in lane P-4.5 (hitchhiker 192.1d2). It cannot be determined a successful PCR because the band is faint. There is one faint band for 197.2a4 DNA at 60C. A clear band 197.2a4 DNA at 55C but not for 60C suggests that 55C is closer to its optimum annealing temperature. There are three bands for primer five for DNAs 197.2a4, hitchhiker 9 and hitchhiker 192.1d2 at 55C. These DNAs also show faint bands at 60C suggesting the annealing temperature was too high for the primers to bind to the template properly. There were no bands whatsoever for looploop DNA and ch2 cDNA for any of the primers at either temperature.

Crc/CBA DNA using primer 1 was chosen in order to continue with the experiment because it was not possible to reattempt the PCRs using primers 3 to 5 without further delaying the project. It was important that the primer in the 3’UTR region was chosen to ensure specificity to Rgnef. 3’UTRs bind RNA-binding proteins to regulate gene expression. The binding proteins bind to 3’UTR-cis-elements to mediate 3’UTR functions by recruiting effector proteins (Baltz et al., 2012). A 350 bp long target of Rgnef DNA was identified for amplification via PCR (Figure 26). Rgnef-E1_Forward bound to complementary regions of the Rgnef coding sequence in positions 4261 to 4280. Rgnef-E1_Reverse bound at positions 331 to 350 on the Rgnef coding sequence. The figure shows the whole region is within an exon; important because the gene product is encoded by exons only. The diagram also shows a miscellaneous feature from position 4591 to 4610 on the target sequence. Miscellaneous features are new or rare regions of biological interest which cannot be described by an alternative feature key (Annotating Your Sequence for Submission, 2011).

Small amounts of crc/CBA DNA were amplified using PCR (Figure 22). The DNA was taken from an elution after a purification process using the QIAquick spin column to determine whether there was DNA still present. Lane 1 shows a band around 350 bp and an intensity equal to 50 ng of PCR product. The RNA band was around ten times more intense than the plasmid band, indicating that around 10 g/l of the probe was synthesised.

Rgnef was inserted into the pGEM®-T Easy vector system and amplified via PCR during a restriction and insertion clone process (Figure 23; Figure 27). The plasmids were cut with different restriction enzymes using non-directional cloning whereby a vector plasmid is digested by a single restriction enzyme. Lane 1 and 2 show a linearised plasmid, cut in both strands with SacI and ApaI, respectively. Both bands are around 3000 bp, which is expected because the length of the plasmid and insert is 3365 bp, Lane 3 shows an uncut plasmid with an unclear, indistinct band ranging between 2000 to 4000 bp. This is possible because uncut plasmids can take up different topological forms, affecting how they run on a gel. For example, the plasmid can be supercoiled. This describes the intertwining of two strands when the ends of a linear DNA molecule are ligated to produce a covalently close circle. This conformation means the plasmid migrates through the gel more quickly than predicted (Higgins and Vologodskii, 2015). The plasmid could have also taken up a nicked form where the supercoiling is released due to a cut in one of the DNA strands. This leaves a larger circular plasmid conformation with a slow motility within the gel (Higgins and Vologodskii, 2015). This might explain the indistinct bands in lanes 4, 7 and 10. Lane 6 shows two differently sized bands. Lane 6 has one band around 3000 bp and one band at around 400 bp (circled, yellow). This is because EcoRI enzyme cuts at two sites, either side of the insert. This creates one band the size of the vector and one band the size of the target region fragment. Lanes 8 to 10 contains a plasmid with the gene for sonic hedgehog (Shh) to be used as a positive control. This is because the expression of Shh within a mouse embryo at later stages of gestation is very well documented and therefore provides comparison for Rgnef gene.

Due to the universal nature of M13, the insert could be in either a sense or antisense orientation. Therefore, the plasmid obtained after ligation and midiprep was sent off for sequencing. A blast search was made from the sequence generated in order to establish the orientation of the Rgnef probe in the vector, either sense or antisense. Blast uses pairwise comparison to determine a base-by-base matched orientation. The blast search showed he insert was orientated in the sense (5’ – 3’) direction using T7 polymerase rather than using Sp6 polymerase in the antisense (3’ – 5’) direction. The DIG labelled probe synthesised SHH in both the sense and antisense orientations (lane 1 and 2). However, Rgnef only synthesised in the anti-sense orientation (lane 4), not the sense orientation (lane 3) (Figure 24).

In situ Hybridisation
Sagittal and transverse sections of mouse embryos were mounted to slides for Rgnef expression analysis. Sagittal plane divides the embryo into left and right sections. A section that runs parallel to the sagittal plane are known as para-sagittal. Transverse plane divides the anterior and posterior parts of the embryo, perpendicular to the sagittal plane. Some parts of sections seem to have detached themselves from the slide. There is staining in the sections which appears to be Rgnef expression.

E10.25 – E10.5
Firstly, SHH stained transverse sections 10.25 to 10.5 days post conception showed staining in the mesencephalic vesicle and the neuroepithelium (Figure 15; Figure 16). There were no results for the Rgnef probe at this age and plane due to limited section availability.

E12.5 – E13.0
There is staining in the caudal lobe of right lung, medullary region of testes and the liver in parasagittal sections of a 12.5 to 13 days post conception stained with Rgnef. There is also staining in the surface layers of the proximal part of the tail, limb structure and the entrance to the nasal cavity (external naris) (Figure 4; Figure 5).

E14.5
Transverse sections of 14.5 days post conception embryos were labelled with Rgnef and Shh. Rgnef stained sections indicated expression in the ependymal layer, around the whole eye structure, primitive nasal cavity, nasal septum and basilar artery. However, staining seems to be concentrated in the epithelial layers of these structures (Figure 1; Figure 2). Other sets of sections stained with Rgnef had prominent staining in the roof of the midbrain and surrounding ependymal layers. However, there was small amounts of staining in the vascular elements of pia mater as well as early elements of choroid plexus associated with pineal recess of third ventricle, the telencephalic vesicle (future lateral ventricle) and the wall of the telencephalic vesicle (Figure 3; Figure 12). There is also staining in the central canal, primary ossification centre within cartilage primordium of the blade of the scapula with superficial layer of periosteal ossification and the primary palate (Figure 14). Sections stained with Shh indicated similar but stronger expression patterns seen in Rgnef sections. For example, there is clear staining in the mantle layer of the section, vascular elements of the pia mater, wall of the telencephalic vesicle, epithelial layers of the dorsal part of the third ventricle, ependymal layers and roof of the midbrain (Figure 14). There also appears to be staining in the ependymal layer, dorsal surface of tongue mass the eye structures. Other areas of the section show darker staining but detachment of the sections from the slides mean it is difficult to determine which anatomical features they are.

E15.5
Parasagittal embryonic sections were stained with Rgnef, 15.5 days post conception. The darkest is staining on the tip of the tongue. There is also staining in the right seventh costal cartilage, distal part of the tail, cartilage primordium of the shaft of third rib with periosteal collar of bone. Moreover, there is staining along the epidermis of the whole embryo section (Figure 10). A further six embryo sections were stained with different probes to provide positive and negative controls. There was conformation of Rgnef staining in the tip of the tongue (Figure 11a; Figure 11b). The sense negatively controlled sections show only background staining, as expected (Figure 11c; Figure 11d). Shh positively controlled sections show staining in the tip of the tongue as well as some inferior segments of the embryo (Figure 11e; Figure 11f). The parasagittal nature of these sections means it is harder to compare nature in which the sections have been stained because they are not taken from exactly the same axis of the embryo.

E16.5
Transverse embryonic sections stained with the Rgnef probe 16.5 days post conception shows there is staining around the in the dorsal (posterior) root ganglion. There is further staining at the ossification within cartilage primordium of neck region of the rib, lumens of left and right ventricle, xiphisternal origin of the diaphragm, cartilage primordium of mid-shift region of the left rib, as well as the epithelial layers of many of the anatomical features including the surface ectoderm (skin) (Figure 6; Figure 7). There is stronger staining in the centre of the the spinal cord in the lower thoracic region for one of the transverse embryonic sections (Figure 7b). Sagittal sections stained with Rgnef probe 16.5 days post conception indicates staining in the lateral extremity of the pituitary, cochlea, Cartilage primordium of coccygeal vertebral body, distal part of the tail, primordia of follicles of vibrissae associated with the upper lip, ventricular zone and right lateral ventricle (Figure 8; Figure 9). There is clear staining in much of the inferior regions of the embryonic section. However, due to detachment of the embryo from the slide, it is difficult to accurately determine the specific stained features. There is also suggestion of Rgnef expression in the epithelial layers of the different anatomical features of the sections.

Discussion
This report has attempted to address the expression pattern of Rgnef in mouse embryonic development from 10.25 to 16.5 days post conception. Little is known about the Rgnef gene, although there is evidence that it is involved in the formation of pathological neuronal cytoplasmic inclusions in degenerating spinal motor neurons along with the contribution to NTDs through associations with the SHH pathway. Identification of specific causes of NTDs associated with Rgnef will facilitate the development of novel experiments that may improve understanding currently available about Rgnef and its deleterious effects on health.

This study measured Rgnef expression within histological section of mouse embryos aged 10.25 to 16.5 days post conception using in situ hybridisation methodologies. This was achieved using riboprobes complementary to the target mRNA species and non-radioactive DIG-labelled RNA probes specific to Rgnef. This allowed comparison to well-known SHH expression. It was determined that Rgnef and SHH have very similar expression patterns in the embryonic sections analysed. The SHH signalling pathway is involved in the essential patterning of midline structures in the brain, spinal cord and forebrain (Garcia et al., 2018), as well as cell growth, specialisation and normal patterning of the body and eye. This is evident in embryo sections 14.5 days post conception stained with SHH. For example, there is clear staining around the developing brain and neural tube structures. This is consistent with previous findings whereby SHH signalling is a regulator of neural progenitors of the rostral neural tube (Garcia et al., 2018), allowing the establishment of definitive brain structures (Fuccillo et al., 2006). Other than neuronal structures, there is SHH expression staining on the dorsal surface of tongue mass. This is consistent with palate and tooth development from 11.5 days post conception, onwards, where the palatal shelves elevate and fuse at the midline to form an intact palate and the teeth undergo epithelial thickening, bud, cap and bell. SHH is a key signalling molecule which increases cell proliferation rate at the site of tooth development (Hardcastle et al., 1998). Furthermore, Rgnef shows very similar expression patterning to this. This is further evidence of the potential relationship between Rgnef and SHH.

Comparatively to SHH, Rgnef presents increased expression on the epithelial layers of structures during morphogenesis of certain features at that time in development. For example, the ependymal layer, around the whole eye structure, primitive nasal cavity, nasal septum and basilar artery. This is especially interesting because Rgnef has been shown to bind focal adhesion kinases associated with cellular adhesion and cellular spreading processes (Miller et al., 2013). This suggests that Rgnef is heavily involved in the morphogenesis and spatial arrangement of different anatomic features at certain points of embryonic development, something that hasn’t necessarily been extensively reported before.

Like most experiments investigating novel ideas, there were limitations. Firstly, there are numerous advantages of using ISH techniques to visualise gene expression. For example, enables use of embryo tissue which is otherwise difficult to obtain, different hybridisations could be completed on the same tissue section and libraries of tissue expression data can be generated and stored for future use. However, it is difficult to identify expression data low in DNA or RNA copies. Further studies would have to be completed in order to establish whether this was the case as staining was present on Rgnef and SHH segments. There were also problems with detachment of sections from the slides. This might be due to how the sections were treated before setting in wax. In order for the sections to adhere to the slides, they are treated with either 3-triethoxysilylpropylamine (TESPA) or poly-L-lysine. This is so the sections do not detach during the washing steps of ISH (Nagy et al., 2007). Reasons for detachment might be non-optimal pH levels or temperature before setting in paraffin wax (Clark, 2008).

There were also problems with the initial PCRs because there were no visible bands. This suggested the primers were not annealing to the target DNA sequence (Figure 17; Figure 18). It was determined dNTPs and Mg2+ concentration were not the issue. Therefore, completing multiple PCRs using different DNA types allowed the conclusion that the DNA initially used was probably degraded. However, primer sets 1 and 2 produced better results than primers 3 to 5. Reasons for primers 3 to 5 not working effectively might include pseudogene amplification or priming of unintended regions (PCR Troubleshooting, n.d.). Even though there were a few limitations with this study, the experimental model was representative due to amount of data collated from Rgnef stained sections. However, it might be beneficial to collect more data from SHH and negative control sections in order to extrapolate the data fully to a wider context.

As expressed before, Rgnef is an extremely understudied gene in terms of its function and potential impact on various pathogeneses including NTDs. This research has found a possible association between Rgnef and SHH pathway. Therefore, further data needs to be collated to determine the exact relationship. This might assess the role Rgnef has in the morphological and spatial arrangement of proliferative cells.

In conclusion, this study has assessed the expression pattern of Rgnef in mouse embryonic development from 10.25 to 16.5 days post conception using ISH techniques. It has established a potential novel relationship between Rgnef and the SHH pathway not described before in recent literature. This will inevitably lead to continuation of studies regarding the impact of Rgnef and the formation of NCIs associated with ALS, but also novel approaches to investigate the role of Rgnef in NTDs during development

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