Tatjana Sauka-Spengler

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets (‘amphioxus’) are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic ∼520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

A gene regulatory network orchestrates neural crest formation

The neural crest is a multipotent, migratory cell population that is unique to vertebrate embryos and gives rise to many derivatives, ranging from the peripheral nervous system to the craniofacial skeleton and pigment cells. A multimodule gene regulatory network mediates the complex process of neural crest formation, which involves the early induction and maintenance of the precursor pool, emigration of the neural crest progenitors from the neural tube via an epithelial to mesenchymal transition, migration of progenitor cells along distinct pathways and overt differentiation into diverse cell types. Here, we review our current understanding of these processes and discuss the molecular players that are involved in the neural crest gene regulatory network.

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Assembling neural crest regulatory circuits into a gene regulatory network.

The neural crest is a multipotent stem cell–like population that gives rise to a wide range of derivatives in the vertebrate embryo including elements of the craniofacial skeleton and peripheral nervous system as well as melanocytes. The neural crest forms in a series of regulatory steps that include induction and specification of the prospective neural crest territory–neural plate border, specification of bona fide neural crest progenitors, and differentiation into diverse derivatives. These individual processes during neural crest ontogeny are controlled by regulatory circuits that can be assembled into a hierarchical gene regulatory network (GRN). Here we present an overview of the GRN that orchestrates the formation of cranial neural crest cells. Formulation of this network relies on information largely inferred from gene perturbation studies performed in several vertebrate model organisms. Our representation of the cranial neural crest GRN also includes information about direct regulatory interactions obtained from the cis-regulatory analyses performed to date, which increases the resolution of the architectural circuitry within the network.

Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest

The neural crest is a multipotent, stem cell-like population that migrates extensively in the embryo and forms a wide array of derivatives, ranging from neurons to melanocytes and cartilage. Analyses of the gene regulatory network driving neural crest development revealed Sox10 as one of the earliest neural crest-specifying genes, cell-autonomously driving delamination and directly regulating numerous downstream effectors and differentiation gene batteries. In search of direct inputs to the neural crest specifier module, we dissected the chick Sox10 genomic region and isolated two downstream regulatory regions with distinct spatiotemporal activity. A unique element, Sox10E2 represents the earliest-acting neural crest cis-regulatory element, critical for initiating Sox10 expression in newly formed cranial, but not vagal and trunk neural crest. A second element, Sox10E1, acts in later migrating vagal and trunk crest cells. Deep characterization of Sox10E2 reveals Sox9, Ets1, and cMyb as direct inputs mediating enhancer activity. ChIP, DNA-pull down, and gel-shift assays demonstrate their direct binding to the Sox10E2 enhancer in vivo, whereas mutation of their corresponding binding sites, or inactivation of the three upstream regulators, abolishes both reporter and endogenous Sox10 expression. Using cis-regulatory analysis as a tool, our study makes critical connections within the neural crest gene regulatory network, thus being unique in establishing a direct link of upstream effectors to a key neural crest specifier.

Histone Demethylase JmjD2A Regulates Neural Crest Specification

The neural crest is a multipotent stem cell-like population that is induced during gastrulation, but only acquires its characteristic morphology, migratory ability, and gene expression profile after neurulation. This raises the intriguing possibility that precursors are actively maintained by epigenetic influences in a stem cell-like state. Accordingly, we report that dynamic histone modifications are critical for proper temporal control of neural crest gene expression in vivo. The histone demethylase, JumonjiD2A (JmjD2A/KDM4A), is expressed in the forming neural folds. Loss of JmjD2A function causes dramatic downregulation of neural crest specifier genes analyzed by multiplex NanoString and in situ hybridization. Importantly, in vivo chromatin immunoprecipitation reveals direct stage-specific interactions of JmjD2A with regulatory regions of neural crest genes, and associated temporal modifications in methylation states of lysine residues directly affected by JmjD2A activity. Our findings show that chromatin modifications directly control neural crest genes in vertebrate embryos via modulating histone methylation.

Gain-and Loss-of-Function Approaches in the Chick Embryo

The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re-established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001). Transient transfection methods have allowed for efficient mis- and overexpression of transgenes. For long-term analyses, retrovirally mediated gene transfer has particular advantage. For short-term experiments, electroporation and adenoviral-mediated gene transfer methods provide transient expression, largely because of the short persistence time of the transgene within the cell. More recently, Tol2 transposon-mediated constructs have been employed, allowing for integration into the genome and prolonged expression of the transgene (Sato et al., 2007), see Chapter 14 by Takahashi et al., this volume). These methods today are routinely used for gain-of-function analysis, to overexpress or ectopically express genes of interest (Arber et al., 1999; Barembaum and Bronner-Fraser, 2007; Bel-Vialar et al., 2002). Loss-of-function experiments are also possible using electroporation of dominant-negative constructs that act as competitive inhibitors (Bel-Vialar et al., 2002; Renzi et al., 2000; Suzuki-Hirano et al., 2005), morpholino antisense oligos (Basch et al., 2006; Kos et al., 2001; Sheng et al., 2003) that block translation or splicing, or constructs expressing small interfering or small hairpin RNAs (siRNAs or shRNAs) (Chesnutt and Niswander, 2004; Das et al., 2006; Katahira and Nakamura, 2003). Electroporation as the most popular method of the transient transfection into the chick embryos. Electroporation of chicken embryos involves application of an electric field to the exposed tissue that transiently disrupts the stability of the cell plasma membrane, creating reversible pores through which nucleic acids or their analogues can be readily transported into the cytosol. The use of this method for transfection into the vertebrate embryos has been facilitated by adapting the voltage parameters and the type and the duration of the electric pulse. By applying several successive square pulses at a very low voltage, with long rest periods in between, one can successfully deliver a DNA construct or another small charged particle into the cytoplasm, with minimal cell death, high efficiency of the uptake and good embryonic survival rate. The size limit of the DNA molecule that can be transfected in such a way is not yet known, though it is more likely that the size limitation in this procedure (if any) lies within the practical problems of cloning large fragments into the plasmid. We routinely overexpress constructs containing 3-4 kb inserts and coharboring a GFP or RFP reporter whose translation is initiated from an internal ribosomal entry site (IRES), thus allowing easy detection of the electroporated cells.

Genetic dissection of the α-globin super-enhancer in vivo

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

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.