Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine ß-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

Making a head: Neural crest and ectodermal placodes in cranial sensory development.

During development of the vertebrate sensory system, many important components like the sense organs and cranial sensory ganglia arise within the head and neck. Two progenitor populations, the neural crest, and cranial ectodermal placodes, contribute to these developing vertebrate peripheral sensory structures. The interactions and contributions of these cell populations to the development of the lens, olfactory, otic, pituitary gland, and cranial ganglia are vital for appropriate peripheral nervous system development. Here, we review the origins of both neural crest and placode cells at the neural plate border of the early vertebrate embryo and investigate the molecular and environmental signals that influence specification of different sensory regions. Finally, we discuss the underlying molecular pathways contributing to the complex vertebrate sensory system from an evolutionary perspective, from basal vertebrates to amniotes.

A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.

An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing ß-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

Retroviral lineage analysis reveals dual contribution from ectodermal placodes and neural crest cells to avian olfactory sensory and GnRH neurons.

The origin of the neurons and glia in the olfactory system of vertebrates has been controversial, with different cell types attributed to being of ectodermal placode versus neural crest lineage, depending upon the species. Here, we use replication incompetent avian (RIA) retroviruses to perform prospective cell lineage analysis of either presumptive olfactory placode or neural crest cells during early development of the chick embryo. Surprisingly, the results reveal a dual contribution from both the olfactory placode and neural crest cells to sensory neurons in the nose and Gonadotropin Releasing Hormone (GnRH) neurons migrating to the olfactory bulb. We also confirm that olfactory ensheathing glia are solely derived from the neural crest. Finally, our results show that neural crest cells and olfactory placode cells contribute to p63 positive cells, likely to be basal stem cells of the olfactory epithelium. Taken together, these finding provide evidence for previously unknown contributions of neural crest cells to some cell types in the chick olfactory system and help resolve previous discrepancies in the literature.