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Alterations in Gene Expression in the Spinal Cord of Mice Lacking Nfix

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Alterations in Gene Expression in the Spinal Cord of Mice Lacking Nfix Matuzelski et al. BMC Res Notes (2020) 13:437 https://doi.org/10.1186/s13104-020-05278-w BMC Research Notes DATA NOTE Open Access Alterations in gene expression in the spinal cord of mice lacking Nfx Elise Matuzelski1, Alexandra Essebier2, Lachlan Harris1, Richard M. Gronostajski3, Tracey J. Harvey1 and Michael Piper1,4* Abstract Objective: Nuclear Factor One X (NFIX) is a transcription factor expressed by neural stem cells within the develop- ing mouse brain and spinal cord. In order to characterise the pathways by which NFIX may regulate neural stem cell biology within the developing mouse spinal cord, we performed an microarray-based transcriptomic analysis of the / spinal cord of embryonic day (E)14.5 Nfx− − mice in comparison to wild-type controls. Data description: Using microarray and diferential gene expression analyses, we were able to identify diferentially / expressed genes in the spinal cords of E14.5 Nfx− − mice compared to wild-type controls. We performed microarray- / / based sequencing on spinal cords from n 3 E14.5 Nfx− − mice and n 3 E14.5 Nfx+ + mice. Diferential gene expression analysis, using a false discovery= rate (FDR) p-value of p < 0.05,= and a fold change cut-of for diferential / expression of > 1.5, revealed 1351 diferentially regulated genes in the spinal cord of Nfx− − mice. Of these, 828 were upregulated,± and 523 were downregulated. This resource provides a tool to interrogate the role of this transcrip- tion factor in spinal cord development. Keywords: Nuclear Factor One, NFIX, Spinal cord, Microarray Objective NFIs also contribute to mouse spinal cord develop- Transcription factors play a central role in promot- ment. Mice lacking either Nfa or Nfb exhibit reduced ing stem cell biology within the developing embryo. For expression of the astrocyte marker GFAP at E18.5 [13]. example, the Nuclear Factor One (NFI) transcription Moreover, NFIA has been proposed to mediate the factors regulate fetal muscle gene expression [1], retinal switch in neural progenitor cell activity, promoting pro- development [2], and dorsal telencephalic formation [3, gression down a glial lineage, rather than the neural line- 4]. NFIX specifcally promotes neural stem cell diferenti- age [13]. NFIA has more recently been shown to interact ation within the developing neocortex, hippocampus and with SOX9 in order to promote the expression of astro- cerebellum [5–8], as well as within the adult forebrain cytic genes, including Apcdd1 and Mmd2 [14]. We have neurogenic niches [9, 10]. Mechanistically, NFIs have also recently shown that Nfx-defcient mice also exhibit been shown to drive diferentiation within the nervous delayed astrocytic diferentiation within the mouse spi- system via the activation of lineage-specifc gene expres- nal cord, with Nfx expression reliant, at least in part, on sion patterns, as well as via the repression of stem cell the activity of NFIB [15]. However, although we analysed self-renewal genes [11, 12]. the expression of NFIX within the developing mouse spi- nal cord, as well as the broad cellular phenotypes of the spinal cord in Nfx−/− mice, we did not perform a global *Correspondence: [email protected] analysis of those genes that were misregulated in Nfx 1 School of Biomedical Sciences, The Faculty of Medicine, The University of Queensland, Brisbane, QLD 4072, Australia mutant mice. To gain a more comprehensive understand- Full list of author information is available at the end of the article ing of the transcriptional landscape controlled by NFIX © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Matuzelski et al. BMC Res Notes (2020) 13:437 Page 2 of 3 within the spinal cord, here we performed microarray all samples using the Afymetrix Expression Console soft- analysis on E14.5 wild-type and Nfx−/− spinal cords, the ware (build 1.4.1.46). Te resulting.CHP fles were then age at which the expression of NFIX by neural progenitor imported into the Afymetrix Transcription Analysis cells lining the spinal central canal is highest [15]. Console (TAC) software and sample fles were compared in diferential gene expression analyses between Nfx−/− Data description and wild-type samples. Algorithm parameters selected Te rationale behind this profling experiment was to included: genome version mm10, one-way between- understand transcriptional changes arising in the devel- subject ANOVA (unpaired) and ANOVA p-value (con- oping spinal cord in the absence of Nfx. To do this, dition pair) < 0.05. Diferential gene expression analyses Nfx−/− mice [16] and their wild-type littermates were were performed using Afymetrix Transcription Analysis used. Tese mice were maintained on a C57Bl/6J genetic Console (TAC) software. Gene expression levels between background. Heterozygous male and female mice were Nfx−/− and wild-type samples were compared using a placed together overnight to produce time-mated litters. one-way between-subject ANOVA (unpaired) and cor- Te presence of a vaginal plug the following day indi- rected for FDR. A p-value < 0.05 denoted a statistically cated a successful mating was likely, and this was desig- signifcant diference in gene expression between sam- nated E0.5. Polymerase chain reactions (PCR) confrmed ples. A fold change cut-of for diferential expression the genotypes of each mouse. PCR primers are available of > ± 1.5 was also used to stratify diferentially expressed on request. Pregnant dams were euthanized by cervical genes. dislocation. Embryos were removed and placed on ice. Table 1 presents data repositories arising from this Whole spinal cords were dissected from E14.5 Nfx−/− work. Te raw sequencing data are available from GEO and wild-type mouse embryos and snap frozen using (Data set 1) [17]. Diferential gene analysis revealed that dry ice. An RNeasy Micro Kit (QIAGEN) was used to 828 genes were upregulated in the E14.5 spinal cord of extract total RNA from these samples, which were then Nfx−/− mice in comparison to controls, whereas 523 diluted to 80 ng/μL and sent to the Ramaciotti Centre genes were downregulated in the spinal cord of Nfx−/− (University of New South Wales, Australia) for micro- mice in comparison to controls (Data set 2) [18]. array analysis on a Mouse Transcriptome Array 2.0ST (Afymetrix). Te Ramaciotti Centre performed sample Limitations quality control at three points in the process; (1) frst- Tis work complements our recently published work cycle cRNA after clean up (spectrophotomoter and Bio- [15], and provides additional, global insights into the analyzer), (2) second-cycle single-stranded cDNA after changes in transcriptional programs evident in the spinal clean up (spectrophotomoter and Bioanalyzer), and (3) cord in the absence of Nfx. While valuable, there are a fragmented and labelled cDNA (Bioanalyzer and gel-shift number of limitations that need to be considered when assay). All the samples passed these checkpoints. For the interpreting these data. Firstly, our analysis of transcrip- Bioanalyzer analyses, all samples reported an RNA integ- tional changes in the spinal cord of Nfx−/− mice was only rity number > 8. For the spectrophotometer analyses, the conducted at one age. Investigating further embryonic OD 260/280 ratio was used to measure the purity of the and postnatal ages would provide more context around RNA; all samples had values ~ 2. Finally, the QC metric the role of NFIX in regulating genesis of the spinal cord. pos_vs_neg_auc value was used to determine the qual- Secondly, although the expression of NFIX is prominent ity of the array data. Values above 0.8 identify the data as within the neural stem cells of the spinal cord at this age within the bounds for the threshold test; all our samples [15], NFIX is also expressed by other cell types within were > 0.91. the spinal cord. As such, interpretation of the data needs Raw microarray data in the form of.CEL fles were to occur with this caveat in mind. Te use of single cell received from the Ramaciotti Centre and initially pro- RNA-sequencing in future could be one way to circum- cessed by running an SST-RMA (gene level) analysis on vent this limitation, and to garner a more nuanced view Table 1 Overview of data fles Label Name of data fle File types (fle extension) Data repository and identifer (DOI or accession number) Data fle 1 Expression data from embryonic mouse spinal cords CEL fle https ://ident ifer s.org/geo:GSE15 5327 [17] from NFIX knockout and wild-type mice Data fle 2 Diferentially regulated genes E14.5 spial cord NFIX Excel spreadsheet (.xlsx) https ://doi.org/10.6084/m9.fgsh are.12735 860.v1 [18] KO vs. WT Matuzelski et al. BMC Res Notes (2020) 13:437 Page 3 of 3 of how NFIX regulates the activity of diferent cell types References 1.
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