Zebrafish pigment cells develop directly from persistent highly multipotent progenitors.

Neural crest cells are highly multipotent stem cells, but it remains unclear how their fate restriction to specific fates occurs. The direct fate restriction model hypothesises that migrating cells maintain full multipotency, whilst progressive fate restriction envisages fully multipotent cells transitioning to partially-restricted intermediates before committing to individual fates. Using zebrafish pigment cell development as a model, we show applying NanoString hybridization single cell transcriptional profiling and RNAscope in situ hybridization that neural crest cells retain broad multipotency throughout migration and even in post-migratory cells in vivo, with no evidence for partially-restricted intermediates. We find that leukocyte tyrosine kinase early expression marks a multipotent stage, with signalling driving iridophore differentiation through repression of fate-specific transcription factors for other fates. We reconcile the direct and progressive fate restriction models by proposing that pigment cell development occurs directly, but dynamically, from a highly multipotent state, consistent with our recently-proposed Cyclical Fate Restriction model.

Trunk Neural Crest Migratory Position and Asymmetric Division Predict Terminal Differentiation.

The generation of complex structures during embryogenesis requires the controlled migration and differentiation of cells from distant origins. How these processes are coordinated and impact each other to form functional structures is not fully understood. Neural crest cells migrate extensively giving rise to many cell types. In the trunk, neural crest cells migrate collectively forming chains comprised of cells with distinct migratory identities: one leader cell at the front of the group directs migration, while followers track the leader forming the body of the chain. Herein we analysed the relationship between trunk neural crest migratory identity and terminal differentiation. We found that trunk neural crest migration and fate allocation is coherent. Leader cells that initiate movement give rise to the most distal derivativities. Interestingly, the asymmetric division of leaders separates migratory identity and fate. The distal daughter cell retains the leader identity and clonally forms the Sympathetic Ganglia. The proximal sibling migrates as a follower and gives rise to Schwann cells. The sympathetic neuron transcription factor phox2bb is strongly expressed by leaders from early stages of migration, suggesting that specification and migration occur concomitantly and in coordination. Followers divide symmetrically and their fate correlates with their position in the chain.

Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such as cancer. How cell cycle progression affects migration, and vice versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish trunk neural crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression.

Activation of the WNT-BMP-FGF Regulatory Network Induces the Onset of Cell Death in Anterior Mesodermal Cells to Establish the ANZ.

The spatiotemporal control of programmed cell death (PCD) plays a significant role in sculpting the limb. In the early avian limb bud, the anterior necrotic zone (ANZ) and the posterior necrotic zone are two cell death regions associated with digit number reduction. In this study, we evaluated the first events triggered by the FGF, BMP, and WNT signaling interactions to initiate cell death in the anterior margin of the limb to establish the ANZ. This study demonstrates that in a period of two to 8 h after the inhibition of WNT or FGF signaling or the activation of BMP signaling, cell death was induced in the anterior margin of the limb concomitantly with the regulation of Dkk, Fgf8, and Bmp4 expression. Comparing the gene expression profile between the ANZ and the undifferentiated zone at 22HH and 25HH and between the ANZ of 22HH and 25HH stages correlates with functional programs controlled by the regulatory network FGF, BMP, and WNT signaling in the anterior margin of the limb. This work provides novel insights to recognize a negative feedback loop between FGF8, BMP4, and DKK to control the onset of cell death in the anterior margin of the limb to the establishment of the ANZ.

Novel generic models for differentiating stem cells reveal oscillatory mechanisms.

Understanding cell fate selection remains a central challenge in developmental biology. We present a class of simple yet biologically motivated mathematical models for cell differentiation that generically generate oscillations and hence suggest alternatives to the standard framework based on Waddington's epigenetic landscape. The models allow us to suggest two generic dynamical scenarios that describe the differentiation process. In the first scenario, gradual variation of a single control parameter is responsible for both entering and exiting the oscillatory regime. In the second scenario, two control parameters vary: one responsible for entering, and the other for exiting the oscillatory regime. We analyse the standard repressilator and four variants of it and show the dynamical behaviours associated with each scenario. We present a thorough analysis of the associated bifurcations and argue that gene regulatory networks with these repressilator-like characteristics are promising candidates to describe cell fate selection through an oscillatory process.

Cyclical fate restriction: a new view of neural crest cell fate specification.

Neural crest cells are crucial in development, not least because of their remarkable multipotency. Early findings stimulated two hypotheses for how fate specification and commitment from fully multipotent neural crest cells might occur, progressive fate restriction (PFR) and direct fate restriction, differing in whether partially restricted intermediates were involved. Initially hotly debated, they remain unreconciled, although PFR has become favoured. However, testing of a PFR hypothesis of zebrafish pigment cell development refutes this view. We propose a novel 'cyclical fate restriction' hypothesis, based upon a more dynamic view of transcriptional states, reconciling the experimental evidence underpinning the traditional hypotheses.

Neural Crest Methodologies in Zebrafish and Medaka.

Neural crest cells are highly multipotent and strongly migratory cells and generate adult neural crest stem cells with varied roles in cellular homeostasis and regeneration. The optical transparency and ready accessibility of fish embryos make them particularly well-suited to high-resolution analysis of neural crest development. However, the dispersive nature of these cells adds to the challenge of their study, requiring that they be identified using marker expression. We describe key protocols for the analysis of neural crest marker expression in zebrafish and medaka, including whole-mount in situ hybridization to detect mRNA using conventional chromogenic substrates and the more recent RNAscope which gives readily multiplexed fluorescent detection and immunofluorescent detection of antigens.

Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependent pigment progenitors in zebrafish.

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk, but also supernumerary cells restricted to the Ventral Stripe. Contrary to expectations, these melanocytes and iridophores are discrete cells, but closely apposed. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels, most prominently in the medial blood vessels, consistent with the ventral trunk phenotype. We provide evidence that neuronal fates are not affected in parade mutants, arguing against transdifferentiation of sympathetic neurons to pigment cells. We show that inhibition of BMP signaling prevents specification of sympathetic neurons, indicating conservation of this molecular mechanism with chick and mouse. However, inhibition of sympathetic neuron differentiation does not enhance the parade phenotype. Instead, we pinpoint ventral trunk-restricted proliferation of neural crest cells as an early feature of the parade phenotype. Importantly, using a chemical genetic screen for rescue of the ectopic pigment cell phenotype of parade mutants (whilst leaving the embryonic pattern untouched), we identify ErbB inhibitors as a key hit. The time-window of sensitivity to these inhibitors mirrors precisely the window defined previously as crucial for the setting aside of APSCs in the embryo, strongly implicating adult pigment stem cells as the source of the ectopic pigment cells. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors.

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these components form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signaling, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unexpected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic phenotype. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the balance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernumerary DRG sensory neurons in a wild-type background, and can rescue the sensory neuron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10(baz1) protein's unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lateral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates.