Natalya Nikitina

Dissecting early regulatory relationships in the lamprey neural crest gene network

The neural crest, a multipotent embryonic cell type, originates at the border between neural and nonneural ectoderm. After neural tube closure, these cells undergo an epithelial–mesenchymal transition, migrate to precise, often distant locations, and differentiate into diverse derivatives. Analyses of expression and function of signaling and transcription factors in higher vertebrates has led to the proposal that a neural crest gene regulatory network (NC-GRN) orchestrates neural crest formation. Here, we interrogate the NC-GRN in the lamprey, taking advantage of its slow development and basal phylogenetic position to resolve early inductive events, 1 regulatory step at the time. To establish regulatory relationships at the neural plate border, we assess relative expression of 6 neural crest network genes and effects of individually perturbing each on the remaining 5. The results refine an upstream portion of the NC-GRN and reveal unexpected order and linkages therein; e.g., lamprey AP-2 appears to function early as a neural plate border rather than a neural crest specifier and in a pathway linked to MsxA but independent of ZicA. These findings provide an ancestral framework for performing comparative tests in higher vertebrates in which network linkages may be more difficult to resolve because of their rapid development.

The sea lamprey Petromyzon marinus: a model for evolutionary and developmental biology.

Sea lampreys (Petromyzon marinus) are cyclostomes, the most basal extant group of vertebrates, and are thought to have existed largely unchanged for more than 500 million years. They are aquatic, eel-shaped animals that spend a major part of their life as filter-feeding larvae called ammocoetes, inhabiting many freshwater bodies in the northern hemisphere. After metamorphosis, sea lampreys migrate to the ocean (or to the Great Lakes), where they feed on the blood and bodily fluids of salmonid fish and ultimately return to freshwater streams and rivers to spawn and die. The unique evolutionary position of lampreys and the relative ease of obtaining mature adults and embryos make this animal an ideal model for investigations into early vertebrate evolution. Studies of features shared between lampreys and jawed vertebrates, but distinct from those in nonvertebrate chordates, have provided information on the origin and evolution of hallmark vertebrate characteristics such as the neural crest, ectodermal placodes, and jaw. In addition, studies of features that are unique to lampreys (e.g., the variable lymphocyte receptor-mediated immune system) provide insights into mechanisms of parallel evolution (e.g., the adaptive immune system). With the establishment of techniques for the extended maintenance and spawning of lampreys in the laboratory, the sequencing of the lamprey genome, and the adaptation and optimization of many established molecular biology and histochemistry techniques for use in this species, P. marinus is poised to become an evolutionary developmental model of choice.

Chapter 1 Gene Regulatory Networks in Neural Crest Development and Evolution

The neural crest is a multipotent migratory embryonic cell population that is present in all vertebrates, but missing from basal chordates. In this chapter, we discuss recent work in amphioxus, ascidians, lamprey, and gnathostomes that reflects the current state of knowledge of the evolutionary origin of this fascinating cell population. We summarize recent evidence for the ongoing diversification of the neural crest in several vertebrate species, with particular reference to studies in nontraditional vertebrate model organisms.

Gene regulatory networks that control the specification of neural-crest cells in the lamprey.

The lamprey is the only basal vertebrate in which large-scale gene perturbation analyses are feasible at present. Studies on this unique animal model promise to contribute both to the understanding of the basic neural-crest gene regulatory network architecture, and evolution of the neural crest. In this review, we summarize the currently known regulatory relationships underlying formation of the vertebrate neural crest, and discuss new ways of addressing the many remaining questions using lamprey as an experimental model.

Ancestral network module regulating prdm1 expression in the lamprey neural plate border.

prdm1 is an important transcriptional regulator that plays diverse roles during development of a wide variety of vertebrate and invertebrate species. prdm1 is required for neural crest specification in zebrafish, but not in mouse embryos. The role of this gene in neural crest formation in other species has not been examined, and its regulation during embryonic development is poorly understood. Here, we investigate the expression pattern, function, and the upstream regulatory inputs into prdm1 during lamprey neural crest development. prdm1 is strongly expressed in the lamprey neural plate border, suggesting a conserved ancestral role of this gene in the neural crest formation. We found that lamprey neural plate border expression of prdm1 is activated by Ap‐2 and Msx, but is independent of Pax3/7 and Zic. Developmental Dynamics 240:2265–2271, 2011.

Culturing lamprey embryos.

Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. This protocol describes how to produce lamprey embryos by collecting sperm and eggs from mature lampreys, performing fertilization, and culturing the embryos through to the desired developmental stage.

Whole-Mount In Situ Hybridization on Lamprey Embryos

Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. This protocol describes an optimized procedure for RNA in situ hybridization in lamprey embryos.

Microinjection of RNA and Morpholino Oligos into Lamprey Embryos

Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. Lamprey embryos are particularly amenable to injection techniques. Like zebrafish and Xenopus embryos, they have double chorions and are resistant to surface-tension-induced rupture when removed from liquid. They can therefore be injected in a dry dish; this eliminates the need to support the embryo while performing injections and makes the procedure very rapid. Also, a single ovulating female can contain up to 100,000 eggs, so the number of injectable embryos per fertilization is not a limiting factor. Finally, the second division lasts for several hours, providing a very large injection window. This protocol describes how to microinject RNA and morpholinos into lamprey embryos for genetic modification studies.

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Immunostaining of Whole-Mount and Sectioned Lamprey Embryos

Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. This protocol describes how to immunostain whole-mount or sectioned lamprey embryos using an antibody raised against the protein of interest and detected with a horseradish peroxidase (HRP)-conjugated secondary antibody.