Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development

Birdsand mammals,phylogeneticallyclose amnioteswith similar postgastruladevelopment,exhibitlittleconservationintheirpost-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We havecarriedoutmorphologicalandcellularanalysesofcleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates(e.g.zebrafishandXenopus).Theincreaseinthenumberof cell layers after EGK-III is not a direct consequence of oriented cell division.Finally,wepresentevidencethat,asinthezebrafishembryo,a yolksyncytiallayerisformedintheavianembryoafterEGK-V.Ourdata suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-toamniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.