Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution Andrew D. Johnson, a,b, * Matthew Drum, b Rosemary F. Bachvarova, c Thomas Masi, b Mary E. White, d and Brian I. Crother d a Division of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK b Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA c Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA d Department of Biological Science, Southeastern Louisiana University, Hammond, LA 70402, USA *Author for correspondence (e-mail: andrew.d.johnson@nottingham.ac.uk) SUMMARY The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos. INTRODUCTION Variations in adult morphology arising during evolution are attributable to changes in developmental processes. The embryos of many vertebrates pass through a morphologically similar stage, the pharyngula, occurring after the completion of neurulation, which is characterized by visible somites, prominent pharyngeal arches, and ventral flexure occurring at the position of the neck (i.e., cervical flexure). Embryo- logical variation occurring after the pharyngula stage plays a major role in diversification at higher order taxonomic levels (Richardson 1999), and understanding the molecular me- chanisms underlying such diversification is currently an area of intense research. However, it is likely that variation of earlier developmental processes, occurring before and during the pharyngula stage, will result in the most pro- found morphological variation, even though these events may also be the most highly constrained and resistant to change (Raff 1996). In this review we consider the evolution of an early developmental process, the germ cell deter- mining mechanisms among vertebrates, and its relation to developmental processes occurring before and after the pharyngula stage. Throughout we use the two major amphibian groups, the anurans (frogs and toads) and the urodeles (salamanders and newts), as the prime examples for discussion, but we also consider several issues at the level of the vertebrates. Anuran and urodele embryos begin to diverge before gastrulation, at least a day before the pharyngula stage. When compared within a phylogenetic context, we note that in all cases early (prepharyngula) anuran development appears to be more derived and shows significant variability among individual species, whereas urodele development retains ancestral features and is less variable within the various urodele lineages that have been examined. Because the germ line develops by very different mechanisms in anuran and urodele embryos, we next considered if this is related to the observed differences in morphogenesis in these groups. EVOLUTION & DEVELOPMENT 5:4, 414–431 (2003) & BLACKWELL PUBLISHING, INC. 414