This conserved period during embryogenesis has been called the phylotypic stage as it represents a point in development of greatest resemblance among members of a phylum (Ballard 1981; Duboule 1994; Hall 1997, 1999; Raff 1996; Sander 1983; Slack et al. 1993; Wolpert 1991). The phylotypic stage has been most thoroughly studied in insects and vertebrates. In insects, this conserved stage was first called Körpergrundgestalt, or the form-building stage by Seidel (1960, as discussed in Hall 1997). Sander (1983) identified this form-building stage with the germ band stage in insects, a larval stage that includes a head, thorax, and abdomen that are already segmented. Consensus among researchers on the timing of the phylotypic stage in vertebrates is not as great as that for insects. Ballard (1981) defines the phylotypic stage as the pharyngula stage, the point in development just following the appearance of the pharyngeal pouches. Slack et al. (1993) suggest that the tailbud stage in vertebrates represents the phylotypic stage. Wolpert (1991) defines the phylotypic stage at the early somite stage following neurulation, whereas Duboule (1994) broadens the stage to encompass the developmental period between the head fold stage and the tailbud stage.
Additionally, researchers noticed that von Baer’s third law (that embryos of different species progressively diverge from one another during ontogeny) does not apply during the earliest stages of development (Elinson 1987; Sander 1983; Seidel 1960). Many examples of early developmental diversity exist, such as the various forms of cleavage and blastula formation in vertebrates, discoidal or spiral cleavage in cephalopods, and the differences between direct and indirect developing echinoderms to name a few (Hall 1996; Raff 1996). Therefore, while many researchers argue that a phylotypic stage does exist, its occurrence is mid-development, and species arrive at this conserved stage via dramatically different routes (Hall 1999; Newman 2011; Raff 1996). This morphologically conserved intermediate stage of development, preceded and succeeded by developmental diversity, has been called the developmental hourglass (Duboule 1994; Hall 1997, 1999; Raff 1996). The hourglass metaphor depicts a constricted middle section, where development is conserved with the wider portions of the hourglass representing greater variation early and late in development (Fig. 3).
Given the difficulties in defining the phylotypic stage in vertebrates, several researchers have questioned its utility in evo-devo (Bininda-Emonds et al. 2003; Richardson 1995; Richardson et al. 1997; Roux and Robinson-Rechavi et al. 2008). Both morphological and molecular studies of embryonic development have led to conflicting results regarding the timing and existence of the phylotypic stage.
For example, after comparing the external embryonic morphologies of 39 species of vertebrates, including representatives from agnathans, cartilaginous fishes, bony fishes, amphibians, reptiles, birds, and mammals, Richardson et al. (1997) concluded that there is no vertebrate phylotypic stage. They noted the challenges of defining a common reference stage that applied to all the species they observed, as common markers used to determine the phylotypic stage differed between the species they studied. The tailbud stage described by Slack et al. (1993) was found to be a promising candidate for the phylotypic stage, as it approximates the end of somite segregation in the trunk region, and it was this stage that these authors used for comparisons. However, the anterior structures in marsupials and monotremes are more advanced than other vertebrate species at the tailbud stage. Additionally, somite number varied dramatically at the tailbud stage, ranging from 11 in the Puerto Rican tree frog to over 60 in the blind worm. Tailbud embryos also varied in size, with the scorpion fish measuring only 700 micrometers and the mudpuppy measuring 9.25 millimeters. They also noted several examples of heterochrony (differential timing of development) at the tailbud stage. For example, in amniotes, the heart has completed looping at the tailbud stage, whereas in zebrafish it has not yet begun to form. Given the morphological diversity among the vertebrates they studied, Richardson et al. (1997) argued that the phylotypic stage does not exist and should perhaps more appropriately be referred to as the phylotypic period.
Another group looked at variation in the timing of developmental events among vertebrates as a whole, and mammals alone, to test the validity of the hourglass model (Bininda-Emonds et al. 2003). Specifically, they looked at two separate datasets, the first included 41 developmental events from 14 species of vertebrates, and the second included 116 developmental events from 14 mammal species and two amniote outgroups. Developmental events represented developmental transitions such as the first appearance of a structure (e.g., heart primordia) or some morphogenetic movement (e.g., fusion of the neural folds). The authors argued that analysis of developmental timing variation is valid for testing the hourglass model for two main reasons. The first is that variation in developmental timing provides an index of character linkage, as such variation depends on the dissociation of developmental events. The second reason is based on the predicted phenotypic similarities between species during the phylotypic stage, which according to the hourglass model occurs at mid-development. They argue that shifts in developmental timing will produce phenotypic differences, and therefore, if the hourglass model is correct, there should be minimal variation in developmental timing in the middle of the developmental sequence. However, developmental timing variation was greatest at mid-development for both the vertebrate and mammalian datasets, the opposite pattern to that predicted by the developmental hourglass model. Therefore, the authors concluded that vertebrates do not display a phylotypic stage.
Using molecular rather than morphological data, Roux and Robinson-Rechavi (2008) also provided evidence that brings the existence of the vertebrate phylotypic stage and the hourglass model into question. The authors argue that the phylotypic stage can be determined by the degree of developmental constraint as measured by the effects of directed knockout mutations, transgenic insertions, point mutations, and morpholinos. If the hourglass model applies to vertebrate development, then the effects of these mutations will be greatest at mid-development, representing the constricted portion of the hourglass. To test their hypothesis, Roux and Robinson-Rechavi used gene expression data from mice and zebrafish. Gene expression in mice was measured using EST counts for 26 stages of development, and in zebrafish from DNA microarrays over 14 stages of development. Instead of finding the greatest degree of developmental constraint at mid-development, flanked by less constraint earlier and later in development as predicted by the hourglass model, they found that the greatest amount of constraint occurs at the beginning of development. As development proceeds in both zebrafish and mice, the degree of developmental constraint steadily decreases. The authors suggest their results indicate that the hourglass model does not apply to vertebrate development.
Contrary to the three studies discussed above, results from recent molecular studies suggest that the phylotypic stage does exist and that the hourglass model is valid. For example, in the first use of molecular data to test for the phylotypic stage, Hazkani-Covo et al. (2005) found evidence for the hourglass model in mouse embryos. Their dataset was comprised of over 1,500 mouse genes and their human orthologs that are expressed in over 26 stages of embryonic development. They measured the evolutionary divergence between corresponding orthologous proteins under the prediction that orthologs expressed during mid-development will resemble each other more closely than during earlier or later stages (representing the hourglass model). While their results were far from conclusive, they did suggest that there is evidence for the vertebrate hourglass model in mice. Human and mouse ortholog expression was most similar between the first somites stage and the formation of the posterior neuropore. This developmental timeframe approximates the morphologically defined phylotypic stage in vertebrates.
Using a different molecular approach, Irie and Sehara-Fujisawa (2007) also provide evidence for the vertebrate phylotypic stage. The authors evaluated the expression of conserved genes among vertebrates at different stages of development using a mouse model. Under the assumption that strong developmental constraints occur during the phylotypic stage, they predict that genes conserved among vertebrates would be highly constrained during the phylotypic stage. Their results indicated a highly conserved embryonic period at days 8.0–8.5. Morphologically, this period in mice is marked by the appearance of the pharyngeal arches and somites and corresponds to the vertebrate phylotypic stage.
While the search for the phylotypic stage has generally been focused on vertebrates, Kalinka et al. (2010) used Drosophila species to determine if insects display a phylotypic stage. Specifically, they used DNA microarrays to measure genome-wide gene expression in six separate Drosophila species throughout the course of development. They predicted that variation in morphological patterning might be reflected by variation in gene expression. Therefore, they compared the timing of gene expression across all six species. The authors found that the variation in gene expression timing was least around the extended germ band stage, which corresponds to the insect phylotypic stage and supports the developmental hourglass model.
Despite these conflicting results, I agree with Hall (1997) that our current conception of the body plan and phylotypic stage is valid. Hall (1997) suggests that many arguments against the existence of the phylotypic stage are based on heterochronic (or timing) shifts in development, not morphological shifts. He compares the different mechanisms that lead to the phylotypic stage with the different mechanisms that lead to the gastrula among species. That the gastrula exists as a morphological entity is not questioned no matter how gastrulation is accomplished. Thus, Hall (1997) argues that there is no cause to abandon the body plan concept and the phylotypic stage simply because the developmental mechanisms that produce such conserved features differ among organisms.
Additionally, each of the studies discussed above uses different methods and criteria to determine the existence of the phylotypic stage. Comparisons among results are impossible given the differences in the studies. What is clear is that we must separate patterns from processes when we discuss the body plan concept. As Hall (1996) states so succinctly “Baupläne represent fundamental, structural and phylogenetic organization that is maintained despite variation in the developmental processes producing the structures.” That conserved morphological patterns exist is clear. In fact, one of the most extraordinary evolutionary events known—the Cambrian explosion—provides evidence for the existence of body plans and marks the initial appearance of all animal forms.