The vertebrate phylotypic stage and an early bilaterian-related stage in mouse embryogenesis defined by genomic information

Abstract

Background: Embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. This "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. However, much controversy remains about when the phylotypic stage appears, and whether it even exists. To overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag (EST) data, gene ontologies (GO), and available genomes of various animals. If strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage.

Results: We established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. The numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day 8.0-8.5, the time of appearance of the pharyngeal arch and somites. During this period, the mouse prominently expresses GO-determined developmental genes shared among vertebrates. Similar analyses revealed the existence of a bilaterian-related period, during which GO-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. The genes associated with the phylotypic stage identified by our method are essential in embryogenesis.

Conclusion: Our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. Furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. These results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von Baer. Identification of conserved stages and tissues by this method in various animals would be a powerful tool to examine the phylotypic stage hypothesis, and to understand which kinds of developmental events and gene sets are evolutionarily constrained and how they limit the possible variations of animal basic body plans.

Figure 1

Genome datasets used to define the evolutional classifications. The evolutional classifications defined in our analyses are indicated, together with arrows covering the range of their putative evolutional origin (e.g. Vertebrate genes represent the genes that are considered to have already existed before vertebrate evolution). Numbers in parentheses indicate developmental genes and total genes classified in each evolutional classification. These classifications are consistent with a recent report [25, 26] that most of the duplications of developmental genes occurred before bilaterian and vertebrate evolution, supporting the appropriateness of our homologue determination (See Additional file 1: Taxonomic classification of homologues of mouse protein-coding genes according to taxonomic range.). See also Methods for the definition of evolutional classifications and "developmental genes".

Figure 2

Moving group analyses of individual ancestor indexes obtained during embryogenesis. A–E: moving group analysis of the ancestor indexes within all genes (25,613 genes). Grouped stage number represents the earlier stage of a pair of developmental stages (e.g. gene expression of grouped stage 14 represents non-redundant genes expressed at stage 14 or 15; see Table 1 for staging). Each stage contained on average 22,744 EST counts (4,418–44,210), which corresponded to on average 4,396 (1,493–6,507) non-redundant ENSEMBL M. musculus genes. F–G: Moving group analysis of the ancestor indexes within developmental genes. Each stage contained on average 1,479 EST counts (282–3,406), which corresponded to 303 (94–491) non-redundant ENSEMBL M. musculus genes. E, M and L indicate the early, middle and late embryonic periods, covering non-redundant genes expressed in stages 2–7, 11–18 and 25–31, respectively. Statistical analysis was performed with Fisher's exact test. Significant differences are indicated by asterisks. n.s: P > 0.05. Within each graph, relative areas of circles reflect the number of expressed (total or developmental) genes at each stage.