Neural crest cells bulldoze through the microenvironment using water channel proteins to stabilize filopodia

Neural crest migration requires cells to move through dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin-1 (AQP-1) signaling. We find that migrating cranial neural crest cells express AQP-1 mRNA and protein within cell filopodia, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed, direction, and the length and stability of cell filopodia. Further, AQP-1 enhances matrix metalloprotease (MMP) activity and phosphorylated focal adhesion kinases (pFAK). Co-localization of AQP-1 expression with EphB guidance receptors in the same migrating neural crest cells raises novel implications for the concept of guided bulldozing by lead cells during migration. Cytometry, RNA-seq and Analysis: Premigratory neural crest were electroporated with either AQP-1 FL or pMES and eggs re-incubated for 24hrs. The neural crest stream adjacent in rhombomere 4 was isolated from healthy ~HH15 embryos. Five pMES transfected embryos were pooled for each of the 3 biological replicates (n=15 total). Four AQP-1 FL embryos were pooled for each of the 3 biological replicates (n=12 total). Tissue was dissociated as previously described (McLennan et al., 2015). Cells were isolated by FACS, which included forward scatter, side scatter, pulse width, live/dead stain (7AAD) and YFP gates as previously described (McLennan et al., 2015; Morrison et al., 2017). Cells were sorted directly into 7ul of Clontech lysis solution containing 0.05% RNase inhibitor. Following lysis for 5 minutes at room temperature, lysates were immediately frozen on dry ice and stored at -80C. Bulk RNA-seq lysates were thawed on ice. cDNA synthesis and library preparation were performed with SMART-seq v4 Ultra Low Input RNA-seq (634891, Takara, Kusatsu, Shiga, Japan) and Nextera XT DNA sample prep and indexing library preparation kits as recommended by the manufacturer (FC-131-2001, FC-131-2004, and FC-131-1096, Illumina, San Diego, CA). Resulting short fragment libraries were checked for quality and quantity using a Bioanalyzer (Agilent) and Qubit Fluorometer (Life Technologies). Libraries were pooled, re-quantified and sequenced as 50bp single reads on 2 lanes of the Illumina HiSeq 2500 in High Output mode using HiSeq Control Software v2.2.58 (Illumina). Following sequencing, Illumina Primary Analysis version RTA v1.18.64 and bcl2fastq2 v2.18 were run to demultiplex reads for all libraries and generate FASTQ files. More than 3 million total alignments were produced per sample. Single end 51-base reads were aligned to the chicken genome galGal4 from UCSC with annotations from Ensembl 84 using STAR (2.5.2b) with options –alignEndsType EndToEnd and sjdbScore 2. Downstream analysis was done in R (3.4.1). Differential expression analysis was performed using the edgeR package (3.18.1). Genes were denoted as differentially expressed if they had a p-value less than .05 and a fold change greater than 1.5-fold (absolute value). Gene ontology enrichment was done using a hypergeometric test on lists of differentially expressed genes.