Evaluation of Developmental Toxicants and Signaling Pathways in a Functional Test Based on the Migration of Human Neural Crest Cells

Background: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the U.S. Environmental Protection Agency and others. Because impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC cells is desirable for a DT test battery. Objective: We developed a robust and widely applicable human-relevant NC function assay that would allow for sensitive screening of environmental toxicants and defining toxicity pathways. Methods: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC cells (MINC assay), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways. Results: Methylmercury (50 nM), valproic acid (> 10 µM), and lead-acetate [Pb(CH3CO2)4] (1 µM) affected the migration of NC cells more potently than migration of other cell types. The MINC assay correctly identified the NC toxicants triadimefon and triadimenol. Additionally, it showed different sensitivities to various organic and inorganic mercury compounds. Using the MINC assay and applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC cells. Conclusions: The MINC assay faithfully models human NC cell migration, and it reveals impairment of this function by developmental toxicants with good sensitivity and specificity.

page 7 Detailed list of chemicals and growth factors used in this study • Supplemental Material, Table S3 page 8 Significantly overrepresented GOs associated with migration identified by whole genome expression analysis of neural crest cells relative to hESC • Supplemental Material, Table S4 page 9 Significantly overrepresented GOs associated with migration identified by whole genome expression analysis of neural crest relative to NEP • Supplemental Material, Figure S1 page 10 Measurement of NC migration with a scratch repopulation assay • Supplemental Material, Figure S2 page 11 Pharmacological modulation of NC migration • Supplemental Material, Figure S3 page 12 Actin dynamics in migrating NC and response to a migration accelerating media supplement • Supplemental Material, Figure S4 page 13 Integrin expression in NC and NEP • Supplemental Material, Videos S1, S2, S3 page 13 • Supplemental References page 14

Cell culture of cell lines
The HeLa229 (ATCC number: CCL-2.1), MCF-7 (ATCC number: HTB-22), HEK293 (ATCC number: CRL-1573) and 3T3 (ATCC number: CCL-92) cell lines were cultured in DMEM supplemented with 10% FCS and 2 mM GlutaMax at 37°C in a humidified atmosphere containing 5% CO 2 . Cells were routinely passaged 3 times a week. The migration assay using these cell types was performed essentially as described for NC cells.

Flow cytometry analysis
For flow cytometry analysis, cells were detached using Accutase (PAA) and stained with HNK1 and p75 specific antibodies for 30 min on ice. After incubation with the appropriate secondary antibodies for 30 min on ice, cells were analyzed using an Accuri C6 flow cytometer (Accuri Cytometers, Inc. Ann Arbor, MI USA). Data were processed and analyzed using the Accuri CFlow Plus software.

Microarray labelling and hybridization
For global transcriptional profiling, the total RNA was isolated from neural progenitor cells using Trizol (Invitrogen, Damstadt, Germany), and purified with Qiagen RNeasy mini kits (Qiagen, Hilden, Germany). On column DNase digestion was performed as per the manufacturer's protocol. Before microarray analysis, the RNA was quantified with a NanoDrop N-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and the integrity of RNA was confirmed with a standard sense automated gel electrophoresis system (Experion, Bio-Rad, Hercules, CA, USA). The samples were used for microarray analysis when the RNA quality indicator (RQI) number was higher than 8. For RNA amplification and biotin labelling, 100 ng total RNA were amplified for 16 h with Genechip 3' IVT Express Kit. After amplification, aRNA was purified with magnetic beads, and 15 μg of aRNA were fragmented with fragmentation buffer as per the manufacturer's instructions. 12.5 μg fragmented aRNA were hybridized with Affymetrix Human Genome U133 plus 2.0 arrays as per the manufacturer's instructions. The chips were placed in a GeneChip Hybridization Oven-645 for 16 h at 60 rpm and 45 ºC. For staining and washing, Affymetrix HWS kits were used on a Genechip Fluidics Station-450. For scanning, the Affymetrix Gene-Chip Scanner-3000-7G was used, and the image and quality control assessments were performed with Affymetrix GCOS software. All reagents and instruments were acquired from Affymetrix (Affymetrix, Santa Clara, CA, USA).

Statistical filtration of significantly expressed genes
Robust Multi-array Analysis was used for background correction and normalization. The raw dataset was transformed by Quantile normalization (Bolstad et al. 2003) with the R (Affy)package (Gautier et al. 2004). MAS5 Expression Summary (Pepper et al. 2007) was used to detect present calls. Only 31567 probe sets out of 54613 received present calls as defined by the detection p-value of ≤ 0.05. Probe sets with "present" calls were selected and those with "absent" calls were eliminated. One way Anova calculation was performed considering 'differentiation' as a factor with hESC as the defined control group. Moderated t-test calculation was applied for pairwise comparisons of NEP vs. hESC and NC vs. hESC. Differentially expressed transcripts were filtered with an FDR -controlled P value of ≤ 0.05 (95% confidence interval). A second filter selected for fold-change values. The Benjamini-Hochberg method was used to adjust the raw p-values to multiple testing and to reduce the false discovery rate. Principal component (PC) analysis was performed using the Stats package in R. The first PC axis accounted for 37.4% of the variance in the data set of variable transcripts and the second accounted for 21.1%. All microarray raw data and results have been deposited in a public database (GEO).
[reference number to be added after manuscript acceptance]

Retrieving information on genes belonging to individual Gene Ontologies (GOs)
GOs often consist of 1000 genes or more. It is therefore difficult to display the genes of all Gene Ontologies analyzed in this study. We therefore, provide an easy web based approach to retrieve this information from an online database. The detailed procedure is described below.
Step 1: Open the webpage http://www.ensembl.org/index.html in your webbrowser Step 2: Select BioMart in the top row of links Step 3: Click on Dataset on the left side of the webpage Step 4: Choose the "Ensembl genes 66" Database from the drop down menu Step 5: Choose your Dataset of interest from the drop down menu. In this case "Homo sapiens genes" Step 6: Now click on Filters (found on the left side of the webpage below "Dataset") Step 7: Now expand "Gene Ontology" Step 8: Paste your GO term number of interest to the box "GO Term Accession". Alternatively you can use the respective GO term name and paste it into the box "GO Term Name" Step 9: Now click on "Attributes" (found on the left side of the webpage below "Filters") Step 10: Expand "Gene" and choose which attributes you want to display. We recommend adding "Ensembl Gene ID", "Description" and "Associated Gene Name".
Step 11: Click on "Results" (located top left of the page) Step 12: To remove potential duplicates within the list of genes, check the box "unique results only" Step 13: Using the "View" dropdown menu, you can select the number of genes which are displayed. To see all the genes included in your GO of interest choose "All".

Live cell video imaging of cell migration
Cells were seeded on 35 mm petri dishes (Ibidi GmbH, Munich, Germany) and treated as described above. Phase-contrast images from multiple predefined points (ROI) along the scratch were taken every 5 minutes for 48 h using a Nikon Biostation IM (Nikon GmbH, Duesseldorf, Germany) equipped with a 20x lens. Images were further processed and combined to video files using ImageJ. The width of an image frame is 240 µm.

Statistics and data mining
For the migration assay, the number of migrated cells was manually counted in ≥ 4 different fields per experiment. The untreated control fields contained 150 ± 44 (mean ± SD) migrated cells per field. In 13 independent experiments 672 ± 118 (means ± SEM) cells were counted for the untreated controls. All data displayed are means from independent biological experiments. Each biological experiment consisted of at least 3 technical replicates. Statistical differences were tested with GraphPad Prism 5.0 (Graphpad Software, La Jolla, USA) by applying ANOVA using Bonferroni's post-hoc test. Independent biological experiments (not technical replicates) were the basic unit used for statistical testing.
Supplemental Material, The highest non-cytotoxic concentration, determined in a cell viability assay (resazurin reduction) after 48 h, was used as highest concentration for the NC cell migration assay. The high concentration indicated in the table corresponds to the highest non-cytotoxic concentration found in the pre-screening assay. Where concentration ranges are given for mercurial compounds or lead, the LOEL in the NC migration assay is indicated in brackets. Note the 10 -50 fold differences between organic and inorganic mercury compounds. Whole genome mRNA expression in neural crest cells was analyzed using the affymetrix microarray platform. Gene expression was compared to gene expression in undifferentiated human embryonic stem cells. Significantly upregulated genes in NC cells were then further analyzed using the web-based gene onthologie (GO) analyzing tool g:Profiler (Reimand et al. 2007). Statistically overrepresented GOs dealing with cell migration are displayed. To display the genes belonging to each GO, the procedure described in the Supplemental method section allows easy access to this information.

Measurement of NC migration with a scratch repopulation assay
In a homogenous NC culture, cells were removed mechanically along an about 0.5 mm wide line using a pipette tip. Cells were visualized using the  Methylmercury (grey bars) inhibited both the normal migration (without AlbuMax ® ) and the accelerated migration (with AlbuMax ® ) to the same extent (inhibition by 50 nM methylmercurychloride in the absence of AlbuMax ® was about 57% compared to untreated cells. In the presence of AlbuMax ® , NC cell migration was reduced by about 60%). Data are displayed as means ± SD of 2 independent biological experiments, each performed in triplicates. Data were normalized to untreated controls without AlbuMax ® . **: p < 0.01, ***: p < 0.001.