Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

Abstract

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

“…The fscn1a MZ mutants were previously described [20]. Transgenic lines used were described previously: Tg(sox10 : rfpmb) [22], Tg(sox10 : gfp) [23], Tg(ngn1 : gfp) [24], Tg(phox2b : gfp) [25].…”

“…pCS2- fscn1a was described previously [20, 26]. To generate a non-targeted fscn1a mRNA for rescue experiments with the fscn1a-ATGMO , wild-type zebrafish fscn1a was amplified with SuperScript one-step RT-PCR (Invitrogen) using a forward primer containing 7 silent base pair mismatches: fscn1a-F(mut) - ATGACcGCtAAtGGtACtAGtGAtA (silent wobble mismatches are denoted in lower case).…”

“…Embryonic protein lysates were prepared as previously described [20]. Antibodies used in this study were rabbit anti-FSCN1 (1:2000, Sigma) and mouse anti-GAPDH (1:1000, Abcam).…”

“…Previously, we demonstrated that maternal/zygotic null mutations in zebrafish fascin1a (fscn1a MZ) , a gene that encodes a filopodia protein called Fascin1, result in partially penetrant defects in the directional migration of a subset of NC cells [20]. Concomitant with abnormal directional NC migration, ~20% of fscn1a MZ embryos display defects in the first (mandibular) arch, but not posterior arches, of the craniofacial skeleton as well as a reduction in NC-derived sympathetic and enteric neurons, but not dorsal root ganglia neurons.…”

Zebrafish germ cells: motility and guided migration

“…The receptor Cxcr4b, while evenly distributed around the cell perimeter [40 ], is activated preferentially at one aspect of the cell, initiating an intracellular signaling cascade that biases the formation of protrusions such as blebs and filopodia (the thin cellular protrusions comprised of actin filaments [41,42]) in the direction of high levels of the chemokine. Filopodia are found at the front of different cell types, including metastatic cells and are important for efficient directional migration for example of cancer cells in vitro [43,44], as well as in in vivo contexts, as demonstrated for blood, neuronal and neural crest cells [45][46][47]. Filopodia that are formed at the front of germ cells exhibit special properties; there, they are greater in number, shorter and rapidly turned over relative to the sides and back of the cells [40 ].…”