Initially (Models 1-9), we did not incorporate ECM degradation (i.e. Last, in support of AQP-1 function, our computational model simulations identified cell speed, filopodia stabilization and ECM degradation as key parameters for controlling neural crest cell invasion (Fig. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Thus, the embryonic neural crest is an attractive in vivo model for studying the function of cell invasion genes in mechanistic detail. These results are consistent with previous data that imply a role for AQP-1 in cell migration and invasion across a wide variety of adult mouse and human cancer cell types (Saadoun et al., 2005; Chen et al., 2012, 2015; Wei and Dong, 2015; Xiong et al., 2017; Cao et al., 2006; Hu and Verkman, 2006; Klebe et al., 2015). This implicates AQP-1 in the integrin-mediated focal adhesion signaling pathways previously shown to be important for neural crest cell migration (Desban and Duband, 1997; Desban et al., 2006; Parsons, 2003). Pearson's coefficient for n=5 embryos was recorded and averaged. First, we discovered that modulation of AQP-1 expression altered neural crest cell motility and invasive ability. Nous utilisons des cookies et des outils similaires pour faciliter vos achats, fournir nos services, pour comprendre comment les clients utilisent nos services afin de pouvoir apporter des améliorations, et pour présenter des annonces. P.M.K. In our computational model (supplementary Materials and methods), we initially quantified changes in the dynamics of cells when we recapitulated the effects of overexpression and downregulation of AQP-1 by modulating relevant model parameters. For immunohistochemistry on in vitro cultures, the same antibodies and protocols were followed as for cryosections and whole-mount embryos; however, the incubation times were shorter. The peer review history is available online at https://dev.biologists.org/lookup/doi/10.1242/dev.185231.reviewer-comments.pdf. 7; Models 2, 3; Movie 5). Thirty-nine neural tubes were exposed to 100 µM AZA, 39 neural tubes were exposed to DMSO (0.05 µl in 1 ml) as control for AZA, 32 neural tubes were exposed to 50 µmol/l GM6001 (Anderson et al., 2006) and 32 neural tubes were exposed to DMSO (1 µl in 1 ml) as control for GM6001. For example, AQP-1 is thought to allow cells to rapidly change cell volume to form thin filopodial protrusions that squeeze in between neighboring cells (Papadopoulos et al., 2008; Verkman, 2009; Karlsson et al., 2013) or to collapse a cell protrusion and cause it to retreat from a repulsive signal (Cowan et al., 2000). By spot counting the number of AQP-1 transcripts per neural crest cell, we found that AQP-1 expression was visible in migrating cranial neural crest cells observed at Hamburger and Hamilton stage (HH) 13 (Hamburger and Hamilton, 1951) en route from rhombomere 4 (r4) to the second branchial arch (BA2) (Fig. AQP-1 perturbation affects the expression and activity of MMPs in migrating cranial neural crest cells when measured by RNA-seq profiling, in-gel zymography and a degradation assay (Fig. In AQP-1 MO-transfected embryos, we found fewer migrating transfected neural crest cells in BA2 compared with control MO-transfected embryos (Fig. (H,I) Neural crest migration 12 h after premigratory neural crest cells were labeled with DiI (red) and transfected with pMES (green, H) or AQP-1 FL (green, I). The spots were set to ≥9 µm in size. Dev Dyn. The dotted lines extend to the most extreme data points not considered outliers, and the outliers are plotted individually as red dots. The integrin channel spots included a diameter of 0.6 µm spot with background subtraction and a quality filter was picked using a statistical upper and lower threshold break in the data. And yet, neural crest cells and the surrounding ectodermal cells go on to generate craniofacial structures as distinct as the beak of a toucan, the tusk of a boar, or the horn of a rhinoceros. Using single cell RT-qPCR and transcriptome profiling, we discovered the enhanced expression of several genes in the most invasive chick cranial neural crest cells, including Aquaporin 1 (AQP-1) (McLennan et al., 2015a; Morrison et al., 2017a). This means that many different systems (neural, skin, teeth, head, face, heart, adrenal glands, gastrointestinal tract) will also have a contribution fron the neural crest cells. P-values were calculated using a standard Student's t-test or paired t-test. (A-C) Expression of AQP-1 mRNA by RNAscope (A) in migrating cranial neural crest cells co-labeled with HNK-1 (B) with the number of AQP-1 transcripts per neural crest cell counted (C). Filopodia number is the number of random directions in which a leader samples the concentration of chemoattractant per time step. (E) Representative tracks of neural crest cells with AQP-1 inhibition (pink) and corresponding DMSO controls (blue). The protocol was slightly modified by heating all the gelatin to 60°C so that there was no issue with room temperature and warm gelatin not mixing fully.

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