Biomolecular condensates in cancer biology.

Understanding the characteristics of cancer cells is essential for the development of improved diagnosis and therapeutics. From a gene regulation perspective, the super-enhancer concept has been introduced to systematically understand the molecular mechanisms underlying the identities of various cell types and has been extended to the analysis of cancer cells and cancer genome alterations. In addition, several characteristic features of super-enhancers have led to the recognition of the link between gene regulation and biomolecular condensates, which is often mediated by liquid-liquid phase separation. Several lines of evidence have suggested molecular and biophysical principles and their alterations in cancer cells, which are particularly associated with gene regulation and cell signaling (" transcriptional" and "signaling" condensates). These findings collectively suggest that the modification of biomolecular condensates represents an important mechanism by which cancer cells acquire various cancer hallmark traits and establish functional innovation for cancer initiation and progression. The condensate model also provides the molecular basis of the vulnerability of cancer cells to transcriptional perturbation and further suggests the possibility of therapeutic targeting of condensates. This review summarizes recent findings regarding the relationships between super-enhancers and biomolecular condensate models, multiple scenarios of condensate alterations in cancers, and the potential of the condensate model for therapeutic development.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Developmental hourglass and heterochronic shifts in fin and limb development.

How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark (Chiloscyllium punctatum). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls.

Animals come in all shapes and sizes. This diversity arose through genetic mutations during evolution, but it is unclear exactly how these variations led to the formation of new shapes. There is increasing evidence to suggest that not all shapes are possible and that variability between animals is limited by a phenomenon known as "developmental constraint". These limitations direct parts of the body towards a specific shape as they develop in the embryo. Therefore, understanding the mechanisms underlying these developmental constraints could help explain how different body shapes evolved. The limbs of humans and other mammals evolved from the fins of fish, and this transition is often used to study the role developmental constraints play in evolution. This is an ideal model as there is already a detailed fossil record mapping this evolutionary event, and data pinpointing some of the genes involved in the development of limbs and fins. But this data is incomplete, and a full comparison between the genes activated in the fin and the limb during embryonic development had not been achieved. This is because most fish used for research have undergone recent genetic changes, making it hard to spot which genetic differences are linked to the evolution of the limb. To overcome this barrier, Onimaru et al. compared genetic data from the developing limbs of mice to the developing fins of the brown-banded bamboo shark, which evolves much slower than other fish. This revealed that although many genes commonly played a role in the development of the fin and the limb in the embryo, the activity of these shared genes was not the same. For example, genes that switched on in the late stages of limb development, switched off in the late stages of fin development. But in the middle of development, those differences were relatively small and both species activated very similar sets of genes. Many of these genes were pleiotropic, which means they have important roles in other tissues and therefore mutate less often. This suggests that the mid-stage of limb development is under the strongest level of constraint. Darwin's theory of natural selection explains that mutations drive evolution. But the theory cannot predict what kinds of new body shapes new mutations will produce. Understanding how the activity levels of different genes affect development could help to fill this knowledge gap. This has potential medical applications, for example, understanding why some genetic changes cause more serious problems than others. This work suggests that mutations in genes that are active during the mid-stage of limb development may have the most serious impact.

Predicting gene regulatory regions with a convolutional neural network for processing double-strand genome sequence information.

With advances in sequencing technology, a vast amount of genomic sequence information has become available. However, annotating biological functions particularly of non-protein-coding regions in genome sequences without experiments is still a challenging task. Recently deep learning-based methods were shown to have the ability to predict gene regulatory regions from genome sequences, promising to aid the interpretation of genomic sequence data. Here, we report an improvement of the prediction accuracy for gene regulatory regions by using the design of convolution layers that efficiently process genomic sequence information, and developed a software, DeepGMAP, to train and compare different deep learning-based models (https://github.com/koonimaru/DeepGMAP). First, we demonstrate that our convolution layers, termed forward- and reverse-sequence scan (FRSS) layers, integrate both forward and reverse strand information, and enhance the power to predict gene regulatory regions. Second, we assessed previous studies and identified problems associated with data structures that caused overfitting. Finally, we introduce visualization methods to examine what the program learned. Together, our FRSS layers improve the prediction accuracy for gene regulatory regions.

The evolutionary origin of developmental enhancers in vertebrates: Insights from non-model species.

How random DNA mutations have established the diverse morphology of extant vertebrates is one of the major challenges in evolutionary biology. Thanks to the recent advancement in DNA sequencing technologies, the genome sequences of many non-model species have been determined, which allows us to address previously inaccessible questions about gene regulatory evolution in vertebrates. In particular, the genome sequences of non-teleost ray-finned fishes and cartilaginous fishes offer clues about when and how vertebrates gained developmental enhancers related to morphological traits that were required for the water-to-land transition. In this review, I examine the evolutionary origin of conserved non-coding elements (CNEs), which often function as tissue-specific developmental enhancers, and discuss how CNEs are related to gene regulatory changes that caused the major morphological transitions of vertebrates.

A de novo transcriptome assembly of the zebra bullhead shark, Heterodontus zebra.

Although cartilaginous fishes have played crucial roles in various fields, including evolutionary biology, marine ecology, bioresources, and aquarium exhibitions, molecular information for these species is poorly available. The present study reports a transcriptome assembly from an embryo of the zebra bullhead shark (Heterodontus zebra), produced by paired-end RNA sequencing. Transcriptome data is generated with a de novo transcriptome assembler, Trinity. Amino acid sequences are predicted from the assemblies, using TransDecoder. Because cartilaginous fishes serve as the outgroup of bony vertebrates, the data would contribute to comparative analyses of a various biological fields. In addition, this study would be useful for conservation biology, such as transcriptome-based population genetics.

Shark genomes provide insights into elasmobranch evolution and the origin of vertebrates.

Modern cartilaginous fishes are divided into elasmobranchs (sharks, rays and skates) and chimaeras, and the lack of established whole-genome sequences for the former has prevented our understanding of early vertebrate evolution and the unique phenotypes of elasmobranchs. Here we present de novo whole-genome assemblies of brownbanded bamboo shark and cloudy catshark and an improved assembly of the whale shark genome. These relatively large genomes (3.8-6.7 Gbp) contain sparse distributions of coding genes and regulatory elements and exhibit reduced molecular evolutionary rates. Our thorough genome annotation revealed Hox C genes previously hypothesized to have been lost, as well as distinct gene repertories of opsins and olfactory receptors that would be associated with adaptation to unique underwater niches. We also show the early establishment of the genetic machinery governing mammalian homoeostasis and reproduction at the jawed vertebrate ancestor. This study, supported by genomic, transcriptomic and epigenomic resources, provides a foundation for the comprehensive, molecular exploration of phenotypes unique to sharks and insights into the evolutionary origins of vertebrates.

Inference of the ancestral vertebrate phenotype through vestiges of the whole-genome duplications.

Inferring the phenotype of the last common ancestor of living vertebrates is a challenging problem because of several unresolvable factors. They include the lack of reliable out-groups of living vertebrates, poor information about less fossilizable organs and specialized traits of phylogenetically important species, such as lampreys and hagfishes (e.g. secondary loss of vertebrae in adult hagfishes). These factors undermine the reliability of ancestral reconstruction by traditional character mapping approaches based on maximum parsimony. In this article, we formulate an approach to hypothesizing ancestral vertebrate phenotypes using information from the phylogenetic and functional properties of genes duplicated by genome expansions in early vertebrate evolution. We named the conjecture as 'chronological reconstruction of ohnolog functions (CHROF)'. This CHROF conjecture raises the possibility that the last common ancestor of living vertebrates may have had more complex traits than currently thought.

A staging table for the embryonic development of the brownbanded bamboo shark (Chiloscyllium punctatum).

BACKGROUND: Studying cartilaginous fishes (chondrichthyans) has helped us understand vertebrate evolution and diversity. However, resources such as genome sequences, embryos, and detailed staging tables are limited for species within this clade. To overcome these limitations, we have focused on a species, the brownbanded bamboo shark (Chiloscyllium punctatum), which is a relatively common aquarium species that lays eggs continuously throughout the year. In addition, because of its relatively small genome size, this species is promising for molecular studies. RESULTS: To enhance biological studies of cartilaginous fishes, we establish a normal staging table for the embryonic development of the brownbanded bamboo shark. Bamboo shark embryos take around 118 days to reach the hatching period at 25°C, which is approximately 1.5 times as fast as the small-spotted catshark (Scyliorhinus canicula) takes. Our staging table divides the embryonic period into 38 stages. Furthermore, we found culture conditions that allow early embryos to grow in partially opened egg cases. CONCLUSIONS: In addition to the embryonic staging table, we show that bamboo shark embryos exhibit relatively fast embryonic growth and are amenable to culture, key characteristics that enhance their experimental utility. Therefore, the present study is a foundation for cartilaginous fish research. Developmental Dynamics 247:712-723, 2018. © 2017 Wiley Periodicals, Inc.

Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In amniote embryos, skeletal muscles in the trunk are derived from epithelial dermomyotomes, the ventral margin of which extends ventrally to form body wall muscles. At limb levels, ventral dermomyotomes also generate limb-muscle precursors, an Lbx1-positive cell population that originates from the dermomyotome and migrates distally into the limb bud. In elasmobranchs, however, muscles in the paired fins were believed to be formed by direct somitic extension, a developmental pattern used by the amniote body wall muscles. Here we re-examined the development of pectoral fin muscles in catsharks, Scyliorhinus, and found that chondrichthyan fin muscles are indeed formed from Lbx-positive muscle precursors. Furthermore, these precursors originate from the ventral edge of the dermomyotome, the rest of which extends towards the ventral midline to form body wall muscles. Therefore, the Lbx1-positive, de-epithelialized appendicular muscle precursors appear to have been established in the body plan before the divergence of Chondrichthyes and Osteichthyes.

Publisher Correction: Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In Fig. 2 of this Article originally published, some erroneous lines appeared on the left side of the images in panels c, e and g. The figure should have appeared as shown below. These errors have now been corrected in all versions of the Article.

The fin-to-limb transition as the re-organization of a Turing pattern.

A Turing mechanism implemented by BMP, SOX9 and WNT has been proposed to control mouse digit patterning. However, its generality and contribution to the morphological diversity of fins and limbs has not been explored. Here we provide evidence that the skeletal patterning of the catshark Scyliorhinus canicula pectoral fin is likely driven by a deeply conserved Bmp-Sox9-Wnt Turing network. In catshark fins, the distal nodular elements arise from a periodic spot pattern of Sox9 expression, in contrast to the stripe pattern in mouse digit patterning. However, our computer model shows that the Bmp-Sox9-Wnt network with altered spatial modulation can explain the Sox9 expression in catshark fins. Finally, experimental perturbation of Bmp or Wnt signalling in catshark embryos produces skeletal alterations which match in silico predictions. Together, our results suggest that the broad morphological diversity of the distal fin and limb elements arose from the spatial re-organization of a deeply conserved Turing mechanism.

A shift in anterior-posterior positional information underlies the fin-to-limb evolution.

The pectoral fins of ancestral fishes had multiple proximal elements connected to their pectoral girdles. During the fin-to-limb transition, anterior proximal elements were lost and only the most posterior one remained as the humerus. Thus, we hypothesised that an evolutionary alteration occurred in the anterior-posterior (AP) patterning system of limb buds. In this study, we examined the pectoral fin development of catshark (Scyliorhinus canicula) and revealed that the AP positional values in fin buds are shifted more posteriorly than mouse limb buds. Furthermore, examination of Gli3 function and regulation shows that catshark fins lack a specific AP patterning mechanism, which restricts its expression to an anterior domain in tetrapods. Finally, experimental perturbation of AP patterning in catshark fin buds results in an expansion of posterior values and loss of anterior skeletal elements. Together, these results suggest that a key genetic event of the fin-to-limb transformation was alteration of the AP patterning network.

Acquisition of the paired fins: a view from the sequential evolution of the lateral plate mesoderm.

The origin of paired fins has long been a focus of both paleontologists and developmental biologists. Fossil records indicate that the first pair of fin-like structures emerged in the body wall of early vertebrates. However, extant agnathan lampreys and hagfishes lack paired fins, and thus it has been difficult to determine the developmental processes underlying the ancestral acquisition of paired fins in vertebrates. Fortunately, recent advances in our knowledge of the developmental mechanisms of the lateral plate mesoderm among different taxa have provided clues for understanding the evolutionary origin of vertebrate paired appendages.

Development and evolution of the lateral plate mesoderm: comparative analysis of amphioxus and lamprey with implications for the acquisition of paired fins.

Possession of paired appendages is regarded as a novelty that defines crown gnathostomes and allows sophisticated behavioral and locomotive patterns. During embryonic development, initiation of limb buds in the lateral plate mesoderm involves several steps. First, the lateral plate mesoderm is regionalized into the cardiac mesoderm (CM) and the posterior lateral plate mesoderm (PLPM). Second, in the PLPM, Hox genes are expressed in a collinear manner to establish positional values along the anterior-posterior axis. The developing PLPM splits into somatic and splanchnic layers. In the presumptive limb field of the somatic layer, expression of limb initiation genes appears. To gain insight into the evolutionary sequence leading to the emergence of paired appendages in ancestral vertebrates, we examined the embryonic development of the ventral mesoderm in the cephalochordate amphioxus Branchiostoma floridae and of the lateral plate mesoderm in the agnathan lamprey Lethenteron japonicum, and studied the expression patterns of cognates of genes known to be expressed in these mesodermal layers during amniote development. We observed that, although the amphioxus ventral mesoderm posterior to the pharynx was not regionalized into CM and posterior ventral mesoderm, the lateral plate mesoderm of lampreys was regionalized into CM and PLPM, as in gnathostomes. We also found nested expression of two Hox genes (LjHox5i and LjHox6w) in the PLPM of lamprey embryos. However, histological examination showed that the PLPM of lampreys was not separated into somatic and splanchnic layers. These findings provide insight into the sequential evolutionary changes that occurred in the ancestral lateral plate mesoderm leading to the emergence of paired appendages.

Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum.

Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum. We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium (LjTbx20, LjTbx4/5, and LjIsl1/2A), establishment of left-right heart asymmetry (LjPitxA), and partitioning of the heart tube (LjTbx2/3A), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A-positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.

Heterochronic shift in Hox-mediated activation of sonic hedgehog leads to morphological changes during fin development.

We explored the molecular mechanisms of morphological transformations of vertebrate paired fin/limb evolution by comparative gene expression profiling and functional analyses. In this study, we focused on the temporal differences of the onset of Sonic hedgehog (Shh) expression in paired appendages among different vertebrates. In limb buds of chick and mouse, Shh expression is activated as soon as there is a morphological bud, concomitant with Hoxd10 expression. In dogfish (Scyliorhinus canicula), however, we found that Shh was transcribed late in fin development, concomitant with Hoxd13 expression. We utilized zebrafish as a model to determine whether quantitative changes in hox expression alter the timing of shh expression in pectoral fins of zebrafish embryos. We found that the temporal shift of Shh activity altered the size of endoskeletal elements in paired fins of zebrafish and dogfish. Thus, a threshold level of hox expression determines the onset of shh expression, and the subsequent heterochronic shift of Shh activity can affect the size of the fin endoskeleton. This process may have facilitated major morphological changes in paired appendages during vertebrate limb evolution.

Identification of four Engrailed genes in the Japanese lamprey, Lethenteron japonicum.

We have isolated four homologs of Engrailed genes from the Japanese lamprey, Lethenteron japonicum, an agnathan that occupies a critical phylogenic position between cephalochordates and gnathostomes. We named these four genes LjEngrailedA, LjEngrailedB, LjEngrailedC, and LjEngrailedD. LjEngrailedA, LjEngrailedB, and LjEngrailedD share a major expression domain in the presumptive midbrain-hindbrain boundary region of the central nervous system, although their levels and timing of expression differed. On the other hand, LjEngrailedC transcripts were in the pharyngeal ectoderm and the ventral ectoderm of the body wall. In addition, LjEngrailedA was expressed in the ventral side of the epibranchial muscle precursors. LjEngrailedD transcripts were seen in the mesodermal cells of the mandibular arch and later in a group of cells responsible for the formation of the upper lip, lower lip, and velum. Our results provide clues to the evolution of these structures as well as a possible scenario for duplication events of Engrailed genes.