Reporting SummaryNature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist. Statistical parametersWhen statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main text, or Methods section). n/a ConfirmedThe exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedlyThe statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable. For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Clearly defined error bars State explicitly what error bars represent (e.g. SD, SE, CI)Our web collection on statistics for biologists may be useful.

The enteric nervous system (ENS) of jawed vertebrates arises primarily from vagal neural crest cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal tract. To gain insight into its evolutionary origin, we examined ENS development in the basal jawless vertebrate, the sea lamprey. Surprisingly, we found no evidence for the existence of a vagally-derived enteric neural crest population in the lamprey. Rather, DiI labeling showed that late-migrating cells, originating from the trunk neural tube and associated with nerve fibers, differentiated into neurons within the gut wall and typhlosole. We propose that these trunk-derived neural crest cells are homologous to Schwann cell precursors (SCPs), recently shown in mammalian embryos to populate post-embryonic parasympathetic ganglia1,2, including enteric ganglia3. Our results suggest that neural crest-derived SCPs made an important contribution to the ancient ENS of early jawless vertebrates, a role that was largely subsumed by vagal neural crest cells in early gnathostomes.

The sea lamprey is a basal, jawless vertebrate that possesses many neural crest derivatives, but lacks jaws and sympathetic ganglia. This raises the possibility that the factors involved in sympathetic neuron differentiation were either a gnathostome innovation or already present in lamprey, but serving different purposes. To distinguish between these possibilities, we isolated lamprey homologues of transcription factors associated with peripheral ganglion formation and examined their deployment in lamprey embryos. We further performed DiI labeling of the neural tube combined with neuronal markers to test if neural crest-derived cells migrate to and differentiate in sites colonized by sympathetic ganglia in jawed vertebrates. Consistent with previous anatomical data in adults, our results in lamprey embryos reveal that neural crest cells fail to migrate ventrally to form sympathetic ganglia, though they do form dorsal root ganglia adjacent to the neural tube. Interestingly, however, paralogs of the battery of transcription factors that mediate sympathetic neuron differentiation (dHand, Ascl1 and Phox2b) are present in the lamprey genome and expressed in various sites in the embryo, but fail to overlap in any ganglionic structures. This raises the intriguing possibility that they may have been recruited during gnathostome evolution to a new function in a neural crest derivative.

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution from reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

The identification of a stem cell regulatory gene which is aberrantly expressed in glioma and associated with patient survival would increase the understanding of the role of glioma cancer stem cells (GCSCs) in the virulence of gliomas. Interrogating the genomes of over 4000 brain cancers we identified ZEB1 deletion in ~15% (grade II and III) and 50% of glioblastomas. Meta-analysis of ZEB1 copy number status in 2,988 cases of glioma revealed disruptive ZEB1 deletions associated with decreased survival. We identified ZEB1 binding sites within the LIF (stemness factor) promoter region, and demonstrate LIF repression by ZEB1. ZEB1 knockdown in GCSCs caused LIF induction commensurate with GCSC self-renewal and inhibition of differentiation. IFN-γ treatment to GCSCs induced ZEB1 expression, attenuating LIF activities. These findings implicate ZEB1 as a stem cell regulator in glioma which when deleted leads to increased stemness, tumorigenicity and shortened patient survival.

Members of the Sox (Sry-related high mobility group box) family of transcription factors play a variety of roles during development of both vertebrates and invertebrates. A marked expansion in gene number occurred during emergence of vertebrates, apparently via gene duplication events that are thought to have facilitated new functions. By screening a macroarrayed library as well as the lamprey genome, we have isolated genes of the Sox B, D, E and F subfamilies in the basal jawless vertebrate, lamprey. The expression patterns of all identified Sox genes were examined from gastrulation through early organogenesis (embryonic day 4–14), with particular emphasis on the neural crest, a vertebrate innovation. Coupled with phylogenetic analysis of these Sox genes, the results provide insight into gene duplication and divergence in paralog deployment occurring during early vertebrate evolution.

No abstract