Figure 5. HtrA1 overexpression and SerpinE2 knockdown inhibit cranial neural crest cell migration and adhesion to fibronectin in vitro.

(A) Scheme of migration experiment. The cranial neural crest was explanted from uninjected or injected embryos at stage 17 and cultured on a fibronectin-covered plastic plate. (B–E’’’) Time-lapse of cell migration in CNC explants after culturing for 0, 4, or 7 hr. Note collective cell migration (filled arrowheads) in uninjected controls and explants injected with control-MO, whereas HtrA1 mRNA and SerpinE2-MO block migration (open arrowheads). In B’’–E’’, the surface areas of explants at 0 hr (blue) and 4 hr (red) were determined by ImageJ and superimposed. Scale bar, 0.2 mm. (F) Quantification of initial CNC migration. Indicated is the surface ratio of explants 4 hr versus 0 hr after plating. 12 explants were analyzed per sample. (G) Scheme of adhesion experiment. Upon injection of eGFP mRNA, CNC explants were dissociated in Ca²⁺- and Mg²⁺-free medium, and single cells were cultured on a fibronectin plate. (H–K) Single eGFP-labeled CNC cells after 1 hr culture. Note adhering cells with extended cytoplasmic processes (filled arrowheads) in control sample and after co-injection with control-MO, whereas HtrA1 mRNA and SerpinE2-MO prevent adhesion causing injected cells to acquire a round phenotype (open arrowheads). Scale bar, 0.02 mm. (L) Quantification of CNC adhesion. Indicated is the ratio of adherent cells relative to the control. Analysis of n>1600 cells from at least six explants per sample. CNC, cranial neural crest; eGFP, enhanced green fluorescent protein. Embryos were injected with 100 pg mRNAs and 40 ng MOs. Data in all graphs are displayed as mean ± SD, n = 2; **p<0.01, ****p<0.0001, unpaired t-test.

Figure 5—video 1. Collective migration of neural crest (NC) cells in vitro.

Download video file ^{(509.4KB, mp4)}

Embryos were injected into the animal pole at the four-cell stage with 40 ng standard control morpholino oligonucleotide. The cranial NC was explanted at stage 16/17, plated on fibronectin and imaged 7 hr after culture. Filming was done for 20 min. Note that cells at the leader front of the cluster extend lamellipodia and filopodia. Scale bar, 10 μm.

Figure 5—video 2. Knockdown of SerpinE2 prevents adhesion and migration of neural crest (NC) cells.

Download video file ^{(997.7KB, mp4)}

Embryos were injected into the animal pole at the four-cell stage with 40 ng SerpinE2 morpholino oligonucleotides. The cranial NC was explanted at stage 16/17, plated on fibronectin and imaged 7 hr after culture. Filming was done for 10 min. Note that cells acquire a ball-like shape and freely float around. Scale bar, 10 μm.