An Updated Staging System for Cephalochordate Development: One Table Suits Them All

Abstract

Chordates are divided into three subphyla: Vertebrata, Tunicata, and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata and Tunicata. Lancelets are small, benthic, marine filter feeders, and their roughly three dozen described species are divided into three genera: Branchiostoma, Epigonichthys, and Asymmetron. Due to their phylogenetic position and their stereotypical chordate morphology and genome architecture, lancelets are key models for understanding the evolutionary history of chordates. Lancelets have thus been studied by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. Surprisingly, however, a universal staging system and an unambiguous nomenclature for developing lancelets have not yet been adopted by the scientific community. In this work, we characterized the development of the European lancelet (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, including an unambiguous stage nomenclature. By tracing growth curves of the European lancelet reared at different temperatures, we were able to show that our staging system permitted an easy conversion of any developmental time into a specific stage name. Furthermore, comparisons of embryos and larvae from the European lancelet (B. lanceolatum), the Florida lancelet (Branchiostoma floridae), two Asian lancelets (Branchiostoma belcheri and Branchiostoma japonicum), and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system could readily be applied to other lancelet species. Although the detailed staging description was carried out on developing B. lanceolatum, the comparisons with other lancelet species thus strongly suggested that both staging and nomenclature are applicable to all extant lancelets. We conclude that this description of embryonic and larval development will be of great use for the scientific community and that it should be adopted as the new standard for defining and naming developing lancelets. More generally, we anticipate that this work will facilitate future studies comparing representatives from different chordate lineages.

Somite Compartments in Amphioxus and Its Implications on the Evolution of the Vertebrate Skeletal Tissues

“…Using carefully staged amphioxus embryos (Carvalho et al, 2021), we found that SPARC was initially expressed in the paraxial mesoderm at the early neurula stage (N1 stage, Figures 1A,B), and then at the N2 stage it was strongly expressed in all the segmented somites, except for the first somite (Figures 1C,D). This pattern is consistent with a previous report (Bertrand et al, 2013).…”

“…Fertilization and subsequent culturing of the embryos were carried out as previously described (Yu and Holland, 2009). Amphioxus embryos were staged according to Carvalho et al (2021). BMP signaling was manipulated by treating embryos with recombinant zebrafish BMP-4 protein (zBMP4, R&D Systems) at 250 ng/ml, as previously described, or by treating with the small-molecular inhibitor, Dorsomorphin (Sigma) (Yu et al, 2007).…”

The Ontology of the Amphioxus Anatomy and Life Cycle (AMPHX)

“…Juveniles range in size from 5-7 mm to 3 cm and adults from 3 to 8 cm. Illustrations from zygote to larva periods have been adapted from Carvalho et al (2021). One of the main aims of the AMPHX is to provide a queryable reference source for information about wild-type amphioxus development and anatomy.…”

“…The AMPHX development ontology is based on in vivo observations [see Carvalho et al (2021) for a morphological description of the different developmental stages] and is also inspired by the staging system proposed by Hirakow and Kajita (1990, 1991, 1994, which we have expanded by adding and redefining some developmental stages. AMPHX thus presents amphioxus development subdivided into ten different periods from zygote to adult (Figure 1 and Table 1).…”

“…These studies are mainly carried out on three or four species of the genus Branchiostoma, but recently also on a species of the genus Asymmetron (Holland and Holland, 2010). For this reason, and in order to standardize the results obtained in different species, we established a universal staging system (Carvalho et al, 2021) as well as an ontology of the development and anatomy of this chordate subphylum. This ontology, AMPHX, is the first one developed for a cephalochordate and follows the example of ontologies previously established for other chordate species (Segerdell et al, 2008;Manni et al, 2014;Van Slyke et al, 2014;Hotta et al, 2020).…”