BackgroundA major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised.ResultsWe have combined in vivo testing of DNA fragments in zebrafish and computational comparative genomics to identify the first regulatory regions of the zebrafish sox10 gene. Both approaches converged on the 3' end of the conserved 1st intron as being critical for spatial patterning of sox10 in the embryo. Importantly, we have defined a minimal region crucial for this function. We show that this region contains numerous binding sites for transcription factors known to be essential in early neural crest induction, including Tcf/Lef, Sox and FoxD3. We show that the identity and relative position of these binding sites are conserved between zebrafish and mammals. A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation. Furthermore, we show that β-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results.ConclusionWe have thus identified two major sites of sox10 regulation in vertebrates and provided evidence supporting a role for at least three factors in driving sox10 expression in neural crest, otic epithelium and oligodendrocyte domains.

SummaryTissue damage leads to a robust and rapid inflammatory response whereby leukocytes are actively drawn toward the wound. Hydrogen peroxide (H2O2) has been shown to be an immediate damage signal essential for the recruitment of these inflammatory blood cells to wound sites in both Drosophila and vertebrates [1, 2]. Recent studies in zebrafish have shown that wound-induced H2O2 is detected by the redox-sensitive Src family kinase (SFK) Lyn within the responding blood cells [3]. Here, we show the same signaling occurs in Drosophila inflammatory cells in response to wound-induced H2O2 with mutants for the Lyn homolog Src42A displaying impaired inflammatory migration to wounds. We go on to show that activation of Src42A is necessary to trigger a signaling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (a member of the CED-1 family of apoptotic cell clearance receptors) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark-mediated signaling axis is homologous to the well-established SFK-ITAM-Syk-signaling pathway used in vertebrate adaptive immune responses. Consequently, our results suggest that adaptive immunoreceptor-signaling pathways important in distinguishing self from non-self appear to have evolved from a more-ancient damage response. Furthermore, this changes the role of H2O2 from an inflammatory chemoattractant to an activator signal that primes immune cells to respond to damage cues via the activation of damage receptors such as Draper.

Neural crest stem cells were first isolated from early embryonic neural crest in the early 1990s, but in the past 5 years, there has been a burst of discoveries of neural crest-derived stem cells from diverse locations. Here, we summarize these data, highlighting the characteristics of each stem cell type. These cells vary widely in the markers they express and the variety of cell types they appear to generate. They occupy diverse locations, but in some cases multiple stem cell types apparently occupy physically proximate niches. To date, few molecular similarities can be identified between these stem cells, although a systematic comparison is required. We note other issues worthy of attention, including aspects of the in vivo behavior of these stem cells, their niches, and their lineage relationships. Together, analysis of these issues will clarify this expanding, but still young, field and contribute to exploration of the important therapeutic potential of these cells. Developmental Dynamics 236:3242-3254, 2007.

SUMMARY Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. Here we report that Drosophila Macroglobulin complement-related (Mcr), a complement orthologue, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program.

Accurate lineage tracing is crucial to understanding of developmental and stem cell biology, but is particularly challenging for transient and highly dispersive cell-types like the neural crest (NC). The authors report in this article a new zebrafish transgenic line Tg(-4725sox10:Cre)(ba74). This line expresses Cre under the control of a well-characterized portion of the sox10 promoter and, by crossing to a floxed-reporter line, the authors show in this article that expression in this line is consistent with those described for GFP reporter lines using the same promoter. Reporter expression is readily detected in patterns consistent with the early expression domains. Thus, the authors see all major groups (pigment, neural, and skeletal) of NC-derived cell-types, as well as cell-types derived from the known non-NC sites of sox10 expression, including otic epithelium and oligodendrocytes. This line provides an invaluable tool for the further study of zebrafish NC development and NC-derived stem cells as well as that of the otic vesicle and oligodendrocytes.

Neural crest cells (NCCs) are highly multipotent stem cells. A long-standing controversy exists over the mechanism of NCC fate specification, specifically regarding the presence and potency of intermediate progenitors. The direct fate restriction (DFR) model, based on early in vivo clonal studies, hypothesised that intermediates are absent and that migrating cells maintain full multipotency. However, most authors favour progressive fate restriction (PFR) models, with fully multipotent early NCCs (ENCCs) transitioning to partially-restricted intermediates before committing to individual fates. Here, single cell transcriptional profiling of zebrafish pigment cell development leads to us proposing a Cyclical Fate Restriction mechanism of NCC development that reconciles the DFR and PFR models. Our clustering of single NCC Nanostring transcriptional profiles identifies only broadly multipotent intermediate states between ENCCs and differentiated melanocytes and iridophores. Leukocyte tyrosine kinase (Ltk) marks the multipotent progenitor and iridophores, consistent with biphasic ltk expression. Ltk inhibitor and constitutive activation studies support expression at an early multipotent stage, whilst lineage-tracing of ltk-expressing cells reveals their multipotency extends beyond pigment cell-types to neural fates. We conclude that pigment cell development does not involve a conventional PFR mechanism, but instead occurs directly and more dynamically from a broadly multipotent intermediate state.

Anaplastic lymphoma kinase (ALK) is an important drug target in many cancers, including lymphoma, neuroblastoma, and lung cancer. Here, we demonstrate proof-of-principle for a novel and inexpensive assay for ALK inhibitor activity and identification in zebrafish. We demonstrate that the human oncogenic ALK fusion, NPM-ALK, drives overproduction of iridophores, a highly visible, shiny pigment cell-type in zebrafish. Treatment with the potent ALK inhibitor, TAE684, fully inhibits production of ALK-dependent iridophores. Using our assay, we test multiple properties of TAE684 in vivo, including efficacy, specificity, and toxicity. We note that TAE684 also inhibits the closely related leukocyte tyrosine kinase (Ltk) that is required for endogenous iridophore development. Similar effects are observed with an independent inhibitor, Crizotinib. Our assay can thus be utilized to identify ALK or LTK inhibitors. Importantly, the natural reflectivity of iridophores lends itself to automation for high throughput assessment of ALK and LTK inhibitor compounds in vivo.

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.