Stability in gene expression and body-plan development leads to evolutionary conservation.

BACKGROUND: Phenotypic evolution is mainly explained by selection for phenotypic variation arising from factors including mutation and environmental noise. Recent theoretical and experimental studies have suggested that phenotypes with greater developmental stability tend to have a constant phenotype and gene expression level within a particular genetic and environmental condition, and this positively correlates with stronger evolutionary conservation, even after the accumulation of genetic changes. This could reflect a novel mechanism that contributes to evolutionary conservation; however, it remains unclear whether developmental stability is the cause, or whether at least it contributes to their evolutionary conservation. Here, using Japanese medaka lines, we tested experimentally whether developmental stages and gene expression levels with greater stability led to their evolutionary conservation. RESULTS: We first measured the stability of each gene expression level and developmental stage (defined here as the whole embryonic transcriptome) in the inbred F0 medaka population. We then measured their evolutionary conservation in the F3 generation by crossing the F0 line with the distantly related Japanese medaka line (Teradomori), followed by two rounds of intra-generational crossings. The results indicated that the genes and developmental stages that had smaller variations in the F0 generation showed lower diversity in the hybrid F3 generation, which implies a causal relationship between stability and evolutionary conservation. CONCLUSIONS: These findings suggest that the stability in phenotypes, including the developmental stages and gene expression levels, leads to their evolutionary conservation; this most likely occurs due to their low potential to generate phenotypic variation. In addition, since the highly stable developmental stages match with the body-plan-establishment stage, it also implies that the developmental stability potentially contributed to the strict conservation of animal body plan.

Potential contribution of intrinsic developmental stability toward body plan conservation.

BACKGROUND: Despite the morphological diversity of animals, their basic anatomical patterns-the body plans in each animal phylum-have remained highly conserved over hundreds of millions of evolutionary years. This is attributed to conservation of the body plan-establishing developmental period (the phylotypic period) in each lineage. However, the evolutionary mechanism behind this phylotypic period conservation remains under debate. A variety of hypotheses based on the concept of modern synthesis have been proposed, such as negative selection in the phylotypic period through its vulnerability to embryonic lethality. Here we tested a new hypothesis that the phylotypic period is developmentally stable; it has less potential to produce phenotypic variations than the other stages, and this has most likely led to the evolutionary conservation of body plans. RESULTS: By analyzing the embryos of inbred Japanese medaka embryos raised under the same laboratory conditions and measuring the whole embryonic transcriptome as a phenotype, we found that the phylotypic period has greater developmental stability than other stages. Comparison of phenotypic differences between two wild medaka populations indicated that the phylotypic period and its genes in this period remained less variational, even after environmental and mutational modifications accumulated during intraspecies evolution. Genes with stable expression levels were enriched with those involved in cell-cell signalling and morphological specification such as Wnt and Hox, implying possible involvement in body plan development of these genes. CONCLUSIONS: This study demonstrated the correspondence between the developmental stage with low potential to produce phenotypic variations and that with low diversity in micro- and macroevolution, namely the phylotypic period. Whereas modern synthesis explains evolution as a process of shaping of phenotypic variations caused by mutations, our results highlight the possibility that phenotypic variations are readily limited by the intrinsic nature of organisms, namely developmental stability, thus biasing evolutionary outcomes.

Theories, laws, and models in evo-devo.

Evolutionary developmental biology (evo-devo) is the study of the evolution of developmental mechanisms. Here, I review some of the theories, models, and laws in evo-devo, past and present. Nineteenth-century evo-devo was dominated by recapitulation theory and archetypes. It also gave us germ layer theory, the vertebral theory of the skull, floral organs as modified leaves, and the "inverted invertebrate" theory, among others. Newer theories and models include the frameshift theory, the genetic toolkit for development, the ABC model of flower development, the developmental hourglass, the zootype, Urbilateria, and the hox code. Some of these new theories show the influence of archetypes and recapitulation. Interestingly, recent studies support the old "primordial leaf," "inverted invertebrate," and "segmented head" theories. Furthermore, von Baer's first three laws may now need to be rehabilitated, and the hourglass model modified, in view of what Abzhanov has pointed out about the maternal-zygotic transition. There are many supposed "laws" of evo-devo but I argue that these are merely generalizations about trends in particular lineages. I argue that the "body plan" is an archetype, and is often used in such a way that it lacks any scientific meaning. Looking to the future, one challenge for evo-devo will be to develop new theories and models to accommodate the wealth of new data from high-throughput sequencing, including single-cell sequencing. One step in this direction is the use of sophisticated in silico analyses, as in the "transcriptomic hourglass" models.

Measuring potential effects of the developmental burden associated with the vertebrate notochord.

The notochord functions primarily as a supporting tissue to maintain the anteroposterior axis of primitive chordates, a function that is replaced entirely by the vertebral column in many vertebrates. The notochord still appears during vertebrate embryogenesis and plays a crucial role in the developmental pattern formation of surrounding structures, such as the somites and neural tube, providing the basis for the vertebrate body plan. The indispensable role of the notochord has often been referred to as the developmental burden and used to explain the evolutionary conservation of notochord; however, the existence of this burden has not been successfully exemplified so far. Since the adaptive value of target tissues appears to result in the evolutionary conservation of upstream structures through the developmental burden, we performed comparative gene expression profiling of the notochord, somites, and neural tube during the mid-embryonic stages in turtles and chicken to measure their evolutionary conservation. When compared with the somites and neural tube, overall gene expression profiles in the notochord showed significantly lower or merely comparable levels of conservation. However, genes involved in inductive signalings, such as the sonic hedgehog (Shh) cascade and the formation of functional primary cilia, showed relatively higher levels of conservation in all the three structures analyzed. Collectively, these results suggest that shh signals are critical as the inductive source and receiving structures, possibly constituting the inter-dependencies of developmental burden.

Derivedness Index for Estimating Degree of Phenotypic Evolution of Embryos: A Study of Comparative Transcriptomic Analyses of Chordates and Echinoderms.

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named "derivedness index" to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.

An irregular hourglass pattern describes the tempo of phenotypic development in placental mammal evolution.

Organismal development is defined by progressive transformations that ultimately give rise to distinct tissues and organs. Thus, temporal shifts in ontogeny often reflect key phenotypic differences in phylogeny. Classical theory predicts that interspecific morphological divergence originates towards the end of embryonic or fetal life stages, i.e. the early conservation model. By contrast, the hourglass model predicts interspecific variation early and late in prenatal ontogeny, though with a phylogenetically similar mid-developmental period. This phylotypic period, however, remains challenging to define within large clades such as mammals. Thus, molecular and morphological tests on a mammalian hourglass have not been entirely congruent. Here, we report an hourglass-like pattern for mammalian developmental evolution. By comparing published data on the timing of 74 homologous characters across 51 placental species, we demonstrated that variation in the timing of development decreased late in embryogenesis--when organ formation is highly active. Evolutionary rates of characters related to this timeframe were lowest, coinciding with a phylotypic period that persisted well beyond the pharyngula 'stage'. The trajectory culminated with elevated variation in a handful of fetal and perinatal characters, yielding an irregular hourglass pattern. Our study invites further quantification of ontogeny across diverse amniotes and thus challenges current ideas on the universality of developmental patterns.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

BACKGROUND: Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. RESULTS: By subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. CONCLUSION: In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

Toward understanding the evolution of vertebrate gene regulatory networks: comparative genomics and epigenomic approaches.

Vertebrates, as most animal phyla, originated >500 million years ago during the Cambrian explosion, and progressively radiated into the extant classes. Inferring the evolutionary history of the group requires understanding the architecture of the developmental programs that constrain the vertebrate anatomy. Here, I review recent comparative genomic and epigenomic studies, based on ChIP-seq and chromatin accessibility, which focus on the identification of functionally equivalent cis-regulatory modules among species. This pioneer work, primarily centered in the mammalian lineage, has set the groundwork for further studies in representative vertebrate and chordate species. Mapping of active regulatory regions across lineages will shed new light on the evolutionary forces stabilizing ancestral developmental programs, as well as allowing their variation to sustain morphological adaptations on the inherited vertebrate body plan.

Pluripotency in the light of the developmental hourglass.

The hourglass model of development postulates divergence in early and late embryo development bridged by a period of developmental constraint at mid-embryogenesis. Recently, molecular support for the hourglass model of development has accumulated, with the emphasis on studies using zebrafish and Drosophila species. Across mammals, the hourglass model and specifically divergence in early development has thus far received little attention. Divergence in mammalian pre-implantation development is particularly interesting because of its potential impact on derivation of pluripotent embryonic stem cells. Here, we review recent findings that support the hourglass model of development. We provide striking examples of variation in key events in mammalian peri-implantation development and their potential consequences for pluripotency of embryonic stem cell lines, including mechanisms of cell signalling and differentiation, gene regulatory networks, X-chromosome inactivation, and epigenetic regulation. The variation in these processes indicates divergence in early mammalian development as was postulated by the hourglass model of development. We discuss the naive and primed states of pluripotency in light of this developmental divergence and their implications for human pluripotent stem cell states.