Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates.

In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or "basal" vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants' various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.

The origin and diversification of the developmental mechanisms that pattern the vertebrate head skeleton.

The apparent evolvability of the vertebrate head skeleton has allowed a diverse array of shapes, sizes, and compositions of the head in order to better adapt species to their environments. This encompasses feeding, breathing, sensing, and communicating: the head skeleton somehow participated in the evolution of all these critical processes for the last 500 million years. Through evolution, present head diversity was made possible via developmental modifications to the first head skeletal genetic program. Understanding the development of the vertebrate common ancestor's head skeleton is thus an important step in identifying how different lineages have respectively achieved their many innovations in the head. To this end, cyclostomes (jawless vertebrates) are extremely useful, having diverged from jawed vertebrates approximately 400 million years ago, at the deepest node within living vertebrates. From this ancestral vantage point (that is, the node connecting cyclostomes and gnathostomes) we can best identify the earliest major differences in development between vertebrate classes, and start to address how these might translate onto morphology. In this review we survey what is currently known about the cell biology and gene expression during head development in modern vertebrates, allowing us to better characterize the developmental genetics driving head skeleton formation in the most recent common ancestor of all living vertebrates. By pairing this vertebrate composite with information from fossil chordates, we can also deduce how gene regulatory modules might have been arranged in the ancestral vertebrate head. Together, we can immediately begin to understand which aspects of head skeletal development are the most conserved, and which are divergent, informing us as to when the first differences appear during development, and thus which pathways or cell types might be involved in generating lineage specific shape and structure.

Embryonic expression of endothelins and their receptors in lamprey and frog reveals stem vertebrate origins of complex Endothelin signaling.

Neural crest cells (NCCs) are highly patterned embryonic cells that migrate along stereotyped routes to give rise to a diverse array of adult tissues and cell types. Modern NCCs are thought to have evolved from migratory neural precursors with limited developmental potential and patterning. How this occurred is poorly understood. Endothelin signaling regulates several aspects of NCC development, including their migration, differentiation, and patterning. In jawed vertebrates, Endothelin signaling involves multiple functionally distinct ligands (Edns) and receptors (Ednrs) expressed in various NCC subpopulations. To test the potential role of endothelin signaling diversification in the evolution of modern, highly patterned NCC, we analyzed the expression of the complete set of endothelin ligands and receptors in the jawless vertebrate, the sea lamprey (Petromyzon marinus). To better understand ancestral features of gnathostome edn and ednr expression, we also analyzed all known Endothelin signaling components in the African clawed frog (Xenopus laevis). We found that the sea lamprey has a gnathsotome-like complement of edn and ednr duplicates, and these genes are expressed in patterns highly reminiscent of their gnathostome counterparts. Our results suggest that the duplication and specialization of vertebrate Endothelin signaling coincided with the appearance of highly patterned and multipotent NCCs in stem vertebrates.

Mesodermal origin of median fin mesenchyme and tail muscle in amphibian larvae.

Mesenchyme is an embryonic precursor tissue that generates a range of structures in vertebrates including cartilage, bone, muscle, kidney, and the erythropoietic system. Mesenchyme originates from both mesoderm and the neural crest, an ectodermal cell population, via an epithelial to mesenchymal transition (EMT). Because ectodermal and mesodermal mesenchyme can form in close proximity and give rise to similar derivatives, the embryonic origin of many mesenchyme-derived tissues is still unclear. Recent work using genetic lineage tracing methods have upended classical ideas about the contributions of mesodermal mesenchyme and neural crest to particular structures. Using similar strategies in the Mexican axolotl (Ambystoma mexicanum), and the South African clawed toad (Xenopus laevis), we traced the origins of fin mesenchyme and tail muscle in amphibians. Here we present evidence that fin mesenchyme and striated tail muscle in both animals are derived solely from mesoderm and not from neural crest. In the context of recent work in zebrafish, our experiments suggest that trunk neural crest cells in the last common ancestor of tetrapods and ray-finned fish lacked the ability to form ectomesenchyme and its derivatives.

A gene expression map of the larval Xenopus laevis head reveals developmental changes underlying the evolution of new skeletal elements.

The morphology of the vertebrate head skeleton is highly plastic, with the number, size, shape, and position of its components varying dramatically between groups. While this evolutionary flexibility has been key to vertebrate success, its developmental and genetic bases are poorly understood. The larval head skeleton of the frog Xenopus laevis possesses a unique combination of ancestral tetrapod features and anuran-specific novelties. We built a detailed gene expression map of the head mesenchyme in X. laevis during early larval development, focusing on transcription factor families with known functions in vertebrate head skeleton development. This map was then compared to homologous gene expression in zebrafish, mouse, and shark embryos to identify conserved and evolutionarily flexible aspects of vertebrate head skeleton development. While we observed broad conservation of gene expression between X. laevis and other gnathostomes, we also identified several divergent features that correlate to lineage-specific novelties. We noted a conspicuous change in dlx1/2 and emx2 expression in the second pharyngeal arch, presaging the differentiation of the reduced dorsal hyoid arch skeletal element typical of modern anamniote tetrapods. In the first pharyngeal arch we observed a shift in the expression of the joint inhibitor barx1, and new expression of the joint marker gdf5, shortly before skeletal differentiation. This suggests that the anuran-specific infrarostral cartilage evolved by partitioning of Meckel's cartilage with a new paired joint. Taken together, these comparisons support a model in which early patterning mechanisms divide the vertebrate head mesenchyme into a highly conserved set of skeletal precursor populations. While subtle changes in this early patterning system can affect skeletal element size, they do not appear to underlie the evolution of new joints or cartilages. In contrast, later expression of the genes that regulate skeletal element differentiation can be clearly linked to the evolution of novel skeletal elements. We posit that changes in the expression of downstream regulators of skeletal differentiation, like barx1 and gdf5, is one mechanism by which head skeletal element number and articulation are altered during evolution.

The evolution of the neural crest: new perspectives from lamprey and invertebrate neural crest-like cells.

The neural crest is an embryonic cell population that gives rise to an array of tissues and structures in adult vertebrates including most of the head skeleton. Because neural crest cells (NCCs), and many of their derivatives, are unique to vertebrates, the evolution of the neural crest is thought to have potentiated vertebrate origins and diversification. However, the lack of clear NCC homologs in invertebrate chordates has made it difficult to reconstruct the evolutionary history of modern NCCs. In this review, the development of NCCs in the basal jawless vertebrate, lamprey, is compared with the development of neural crest-like cells in a range of invertebrates to deduce features of the first NCCs and their evolutionary precursors. These comparisons demonstrate that most of the defining attributes of NCCs are widespread features of invertebrate embryonic ectoderm. In addition, they suggest ancient origins for the neural border domain and chondroid skeletal tissue in the first bilaterian, and show that NCCs must have evolved in a chordate with an unduplicated invertebrate-type genome. On the basis of these observations, a stepwise model for the evolution of NCCs involving heterotopic and heterochronic activation of ancient ectodermal gene programs and new responsiveness to preexisting inducing signals is proposed. In light of the phylogenetic distribution of neural crest-like cells, the deep homology of developmental gene networks, and the central role of evolutionary loss in deuterostome evolution, this article concludes with suggestions for future studies in a broad range of bilaterians to test key aspects of this model. WIREs Dev Biol 2013, 2:1-15. doi: 10.1002/wdev.85 For further resources related to this article, please visit the WIREs website.

Gene regulatory evolution and the origin of macroevolutionary novelties: insights from the neural crest.

The appearance of novel anatomic structures during evolution is driven by changes to the networks of transcription factors, signaling pathways, and downstream effector genes controlling development. The nature of the changes to these developmental gene regulatory networks (GRNs) is poorly understood. A striking test case is the evolution of the GRN controlling development of the neural crest (NC). NC cells emerge from the neural plate border (NPB) and contribute to multiple adult structures. While all chordates have a NPB, only in vertebrates do NPB cells express all the genes constituting the neural crest GRN (NC-GRN). Interestingly, invertebrate chordates express orthologs of NC-GRN components in other tissues, revealing that during vertebrate evolution new regulatory connections emerged between transcription factors primitively expressed in the NPB and genes primitively expressed in other tissues. Such interactions could have evolved by two mechanisms. First, transcription factors primitively expressed in the NPB may have evolved new DNA and/or cofactor binding properties (protein neofunctionalization). Alternately, cis-regulatory elements driving NPB expression may have evolved near genes primitively expressed in other tissues (cis-regulatory neofunctionalization). Here we discuss how gene duplication can, in principle, promote either form of neofunctionalization. We review recent published examples of interspecies gene-swap, or regulatory-element-swap, experiments that test both models. Such experiments have yielded little evidence to support the importance of protein neofunctionalization in the emergence of the NC-GRN, but do support the importance of novel cis-regulatory elements in this process. The NC-GRN is an excellent model for the study of gene regulatory and macroevolutionary innovation.

An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest.

The neural crest (NC) is a vertebrate-specific cell population that exhibits remarkable multipotency. Although derived from the neural plate border (NPB) ectoderm, cranial NC (CNC) cells contribute not only to the peripheral nervous system but also to the ectomesenchymal precursors of the head skeleton. To date, the developmental basis for such broad potential has remained elusive. Here, we show that the replacement histone H3.3 is essential during early CNC development for these cells to generate ectomesenchyme and head pigment precursors. In a forward genetic screen in zebrafish, we identified a dominant D123N mutation in h3f3a, one of five zebrafish variant histone H3.3 genes, that eliminates the CNC-derived head skeleton and a subset of pigment cells yet leaves other CNC derivatives and trunk NC intact. Analyses of nucleosome assembly indicate that mutant D123N H3.3 interferes with H3.3 nucleosomal incorporation by forming aberrant H3 homodimers. Consistent with CNC defects arising from insufficient H3.3 incorporation into chromatin, supplying exogenous wild-type H3.3 rescues head skeletal development in mutants. Surprisingly, embryo-wide expression of dominant mutant H3.3 had little effect on embryonic development outside CNC, indicating an unexpectedly specific sensitivity of CNC to defects in H3.3 incorporation. Whereas previous studies had implicated H3.3 in large-scale histone replacement events that generate totipotency during germ line development, our work has revealed an additional role of H3.3 in the broad potential of the ectoderm-derived CNC, including the ability to make the mesoderm-like ectomesenchymal precursors of the head skeleton.

New perspectives on pharyngeal dorsoventral patterning in development and evolution of the vertebrate jaw.

Patterning of the vertebrate facial skeleton involves the progressive partitioning of neural-crest-derived skeletal precursors into distinct subpopulations along the anteroposterior (AP) and dorsoventral (DV) axes. Recent evidence suggests that complex interactions between multiple signaling pathways, in particular Endothelin-1 (Edn1), Bone Morphogenetic Protein (BMP), and Jagged-Notch, are needed to pattern skeletal precursors along the DV axis. Rather than directly determining the morphology of individual skeletal elements, these signals appear to act through several families of transcription factors, including Dlx, Msx, and Hand, to establish dynamic zones of skeletal differentiation. Provocatively, this patterning mechanism is largely conserved from mouse and zebrafish to the jawless vertebrate, lamprey. This implies that the diversification of the vertebrate facial skeleton, including the evolution of the jaw, was driven largely by modifications downstream of a conversed pharyngeal DV patterning program.

Novel Tfap2-mediated control of soxE expression facilitated the evolutionary emergence of the neural crest.

Gene duplication has been proposed to drive the evolution of novel morphologies. After gene duplication, it is unclear whether changes in the resulting paralogs' coding-regions, or in their cis-regulatory elements, contribute most significantly to the assembly of novel gene regulatory networks. The Transcription Factor Activator Protein 2 (Tfap2) was duplicated in the chordate lineage and is essential for development of the neural crest, a tissue that emerged with vertebrates. Using a tfap2-depleted zebrafish background, we test the ability of available gnathostome, agnathan, cephalochordate and insect tfap2 paralogs to drive neural crest development. With the exception of tfap2d (lamprey and zebrafish), all are able to do so. Together with expression analyses, these results indicate that sub-functionalization has occurred among Tfap2 paralogs, but that neo-functionalization of the Tfap2 protein did not drive the emergence of the neural crest. We investigate whether acquisition of novel target genes for Tfap2 might have done so. We show that in neural crest cells Tfap2 directly activates expression of sox10, which encodes a transcription factor essential for neural crest development. The appearance of this regulatory interaction is likely to have coincided with that of the neural crest, because AP2 and SoxE are not co-expressed in amphioxus, and because neural crest enhancers are not detected proximal to amphioxus soxE. We find that sox10 has limited ability to restore the neural crest in Tfap2-deficient embryos. Together, these results show that mutations resulting in novel Tfap2-mediated regulation of sox10 and other targets contributed to the evolution of the neural crest.

A new mechanistic scenario for the origin and evolution of vertebrate cartilage.

The appearance of cellular cartilage was a defining event in vertebrate evolution because it made possible the physical expansion of the vertebrate "new head". Despite its central role in vertebrate evolution, the origin of cellular cartilage has been difficult to understand. This is largely due to a lack of informative evolutionary intermediates linking vertebrate cellular cartilage to the acellular cartilage of invertebrate chordates. The basal jawless vertebrate, lamprey, has long been considered key to understanding the evolution of vertebrate cartilage. However, histological analyses of the lamprey head skeleton suggest it is composed of modern cellular cartilage and a putatively unrelated connective tissue called mucocartilage, with no obvious transitional tissue. Here we take a molecular approach to better understand the evolutionary relationships between lamprey cellular cartilage, gnathostome cellular cartilage, and lamprey mucocartilage. We find that despite overt histological similarity, lamprey and gnathostome cellular cartilage utilize divergent gene regulatory networks (GRNs). While the gnathostome cellular cartilage GRN broadly incorporates Runx, Barx, and Alx transcription factors, lamprey cellular cartilage does not express Runx or Barx, and only deploys Alx genes in certain regions. Furthermore, we find that lamprey mucocartilage, despite its distinctive mesenchymal morphology, deploys every component of the gnathostome cartilage GRN, albeit in different domains. Based on these findings, and previous work, we propose a stepwise model for the evolution of vertebrate cellular cartilage in which the appearance of a generic neural crest-derived skeletal tissue was followed by a phase of skeletal tissue diversification in early agnathans. In the gnathostome lineage, a single type of rigid cellular cartilage became dominant, replacing other skeletal tissues and evolving via gene cooption to become the definitive cellular cartilage of modern jawed vertebrates.

Evidence for the prepattern/cooption model of vertebrate jaw evolution.

The appearance of jaws was a turning point in vertebrate evolution because it allowed primitive vertebrates to capture and process large, motile prey. The vertebrate jaw consists of separate dorsal and ventral skeletal elements connected by a joint. How this structure evolved from the unjointed gill bar of a jawless ancestor is an unresolved question in vertebrate evolution. To understand the developmental bases of this evolutionary transition, we examined the expression of 12 genes involved in vertebrate pharyngeal patterning in the modern jawless fish lamprey. We find nested expression of Dlx genes, as well as combinatorial expression of Msx, Hand and Gsc genes along the dorso-ventral (DV) axis of the lamprey pharynx, indicating gnathostome-type pharyngeal patterning evolved before the appearance of the jaw. In addition, we find that Bapx and Gdf5/6/7, key regulators of joint formation in gnathostomes, are not expressed in the lamprey first arch, whereas Barx, which is absent from the intermediate first arch in gnathostomes, marks this domain in lamprey. Taken together, these data support a new scenario for jaw evolution in which incorporation of Bapx and Gdf5/6/7 into a preexisting DV patterning program drove the evolution of the jaw by altering the identity of intermediate first-arch chondrocytes. We present this "Pre-pattern/Cooption" model as an alternative to current models linking the evolution of the jaw to the de novo appearance of sophisticated pharyngeal DV patterning.