Division of labor during trunk neural crest development

AbstractNeural crest cells, the migratory precursors of numerous cell types including the vertebrate peripheral nervous system, arise in the dorsal neural tube and follow prescribed routes into the embryonic periphery. While the timing and location of neural crest migratory pathways has been well documented in the trunk, a comprehensive collection of signals that guides neural crest migration along these paths has only recently been established. In this review, we outline the molecular cascade of events during trunk neural crest development. After describing the sequential routes taken by trunk neural crest cells, we consider the guidance cues that pattern these neural crest trajectories. We pay particular attention to segmental neural crest development and the steps and signals that generate a metameric peripheral nervous system, attempting to reconcile conflicting observations in chick and mouse. Finally, we compare cranial and trunk neural crest development in order to highlight common themes

Regulation of actin cytoskeleton architecture by Eps8 and Abi1

BACKGROUND: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. RESULTS: Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. CONCLUSION: Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling

DNA methyltransferase 3b is dispensable for mouse neural crest development.

The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures

<i>DNMT3b</i>-deleted neural crest cells have mild migration defects that recover during cranial gangliogenesis.

<p>Migratory neural crest cells and the cranial ganglia they form were visualized by in situ hybridization for <i>Sox10</i> at E8.5 (A, B), E9.0 (C–H), E9.5 (I–K) and E10 (L–M). (A–H) E8.5–9.0 embryos that were <i>DNMT3b^+/fl</i> (A, C; wildtype; n = 6); <i>Wnt1-cre; DNMT3b^fl/−</i> (B, D; homozygous deleted in neural crest cells; n = 13), <i>DNMT3b^+/−</i> (E, G; whole embryo heterozygous; n = 6), and <i>DNMT3b^−/−</i> (F; H; whole embryo homozygous deleted; n = 5). Despite normal migration initially (A, B), dispersed <i>Sox10</i>-positive cells were apparent dorsally, trailing the organized neural crest streams in the branchial arches (ba; arrows in D, F) and eye (e; arrowheads in D, F) in mutant embryos compared to controls (C, E). Subsequently, neural crest cells coalesce to form the trigeminal ganglia (G, H, brackets). (I–N) At E9.5–10, no difference beyond normal embryonic variation was apparent during cranial gangliogenesis between <i>DNMT3b^+/fl</i> (n = 24), <i>Sox10-cre; DNMT3b^fl/−</i> (n = 6), and <i>Wnt1-cre; DNMT3b^fl/−</i> embryos (n = 13). fn, frontonasal region; g, geniculate; n, nodose; o, otic vesicle; p, petrosal; s, somites; t, trigeminal.</p

<i>Wnt1-cre</i> drives cre expression in premigratory cranial neural crest cells, while <i>Sox10-cre</i> is activated during cranial neural crest migration.

<p><i>Wnt1-cre</i> and <i>Sox10-cre</i> transgenic mice were crossed to mice carrying the <i>R26R-lacZ</i> transgene (<i>R26R</i>). Embryos were harvested at various time points and stained for ß-galactosidase activity (A–C, E, G, H) or processed by whole mount in situ hybridization for <i>Wnt1</i> (D) or <i>Sox10</i> (F). At E8.0, <i>Wnt1-cre</i> expression was detectable in the anterior neural plate (A, B; arrowheads). By E8.5, robust <i>Wnt1-cre</i> expression (C) was apparent in the fore- and midbrain, migratory neural crest cells in the first branchial arch stream (arrowhead), and premigratory neural crest cells in the hindbrain (arc and inset). The <i>Wnt1</i> expression pattern (D) for comparison, showing <i>Wnt1</i> expression in cranial neural folds (arrow) and hindbrain (arc). At E8.75, <i>Sox10-cre</i> activity (E) was strong in the first branchial arch (asterisk), low in neural crest cells entering the first (black arrowhead) and second (white arrowhead) branchial arch streams, and undetectable in trunk premigratory and early migratory neural crest cells (dashed line), although these cells express <i>Sox10</i> (F). At E9.5, <i>Wnt1-cre</i> (G) marked trunk neural crest cells while they were still in the neural tube (arc), while <i>Sox10-cre</i> (H) was not expressed in trunk neural crest cells until migration was well underway (dashed arc). Both transgenes labeled cranial neural crest cells at their destinations. e, eye; o, otic vesicle; s, somite.</p

Premigratory and migratory neural crest cells express DNMT3b protein.

<p>6 somite (s; A) and 10 somite (B) wildtype mouse embryos were harvested, 14 µm transverse frozen sections through the midbrain prepared, and DNMT3b (A, B; A’’, B’’ red) and Sox10 (A’, B’; A’’, B’’ green) protein visualized by immunofluorescence. DNMT3b is abundant in the neural plate, including in Sox10-positive neural crest cells in the neural folds (A; arrowheads). Migratory neural crest cells maintain DNMT3b expression (B, arrowheads). The bright staining at the bottom of the images at both stages is in the foregut.</p