Purines as potential morphogens during embryonic development

Abstract

Components of purinergic signalling are expressed in the early embryo raising the possibility that ATP, ADP and adenosine may contribute to the mechanisms of embryonic development. We summarize the available data from four developmental models-mouse, chick, Xenopus and zebrafish. While there are some notable examples where purinergic signalling is indeed important during development, e.g. development of the eye in the frog, it is puzzling that deletion of single components of purinergic signalling often results in rather minor developmental phenotypes. We suggest that a key step in further analysis is to perform combinatorial alterations of expression of purinergic signalling components to uncover their roles in development. We introduce the concept that purinergic signalling could create novel morphogenetic fields to encode spatial location via the concentration of ATP, ADP and adenosine. We show that using minimal assumptions and the known properties of the ectonucleotidases, complex spatial patterns of ATP and adenosine can be set up. These patterns may provide a new way to assess the potential of purinergic signalling in developmental processes.

Fig. 1

Comparative embryogenesis of the mouse, chick, Xenopus and zebrafish. After fertilization (F), these four vertebrates undergo similar phases during their embryonic life. Species-specific stages are indicated on the figure. Briefly, Mouse embryos will implant (imp) at E4.5, after formation of the blastocyst and separation between the epiblast (or primitive ectoderm), at the origin of the embryo, and the cells which will give rise to the extra embryonic structures, the trophoectoderm and the primitive endoderm. The turning process, at E9, allows the establishment of the dorsal and ventral axis. The organogenesis is followed by the embryonic or foetal growth phase and birth takes place between 18 to 21 days after fertilization, depending on the mouse strain. The cleavage phase of the Chick embryos takes place in the hen oviduct. After laying, gastrulation and neurulation are complete in 2 days and stage HH14 is characterized by the existence of 22 somites. Feather germs appear at stage HH30 and organogenesis and embryo growth continue till hatching 22 days after fertilization. Cleavage of Xenopus embryos will be complete by stage 9, blastula stage and neurulation starts at stage 12.5 and ends at stage 20. First somite is formed at stage 17. Organogenesis is the longest phase, characterized by the hatching of the embryos from their vitelline membrane around stage 25. After stage 45, the tadpole will start feeding and will undergo the metamorphosis phase before becoming an adult frog. Zebrafish embryos display the quickest embryonic life cycle. Cleavage divisions lead to the formation of a blastoderm lying over the yolk, at the sphere stage, 4 h after fertilization. Gastrulation starts 5.5 h after fertilization, at the shield stage and is complete only 4.5 h later. Somite formation and neurulation follow during the segmentation period. Organogenesis then takes place during the pharyngula period, less than 24 h after fertilization, and zebrafish embryo usually hatches 48 to 72 h after fertilization. The term larva is being arbitrarily used after the end of the third day, whether the hatching has taken place or not

Fig. 2

Temporal pattern of expression of the purinergic signalling components in mouse, chick, Xenopus and zebrafish embryos. The extent of expression of purinergic receptors and ectonucleotidases is mapped along the major phases of embryogenesis, cleavage, gastrulation, neurulation and organogenesis and embryo growth that have been normalized in duration for the four model organisms to allow easier comparison. The expression of some of these genes has only been described at one stage (indicated by arrows). EST expression data is indicated by blue dashed lines and corresponding gene names in blue. The expression of mouse genes published in [25] or [26] as part of the embryonic mouse database and atlas of gene expression are indicated by solid blue lines. The murine P2Y1 expression by in situ hybridization is available on the MGI website

Fig. 3

Simulation of diffusion and metabolism of ATP—a complex purinergic morphogenetic field. The simulation (values shown at T = 5 s after beginning) shows that ATP and ADP are locally high in concentration over the first 30–40 μm, but that adenosine peaks at around 60 μm and remains high in concentration up to around 120 μm. Thus a complex pattern is produced where the potential for ATP/ADP actions is bounded and limited by a zone where adenosinergic actions have the potential to predominate. The simulation considers a short strip of cells (inset) where one cell at the end releases ATP; the ATP is then converted successively through the intervening intermediates to adenosine, with feed-forward inhibition of the final step mediated by ATP and ADP (inset top). All the metabolites diffuse along the strip with the same diffusion coefficient (300 μm²/s). The conversion of ATP and metabolites are described by Michaelis–Menten kinetics, based on literature values [–95]: ATP, K_m 33.3 μM, V_max, 100 μM/s; ADP, K_m 9.5 μM, V_max 20 μM/s; AMP, K_m 0.94 μM, V_max, 20 μM/s; both ATP and ADP inhibit the conversion of AMP to adenosine (Ado) with a k_i of 0.1 μM. The simulation is based on the following parallel linked equations: ∂[ATP]/∂t = D ∂²[ATP]/∂x ² − k₁[ATP]; ∂[ADP]/∂t = D ∂²[ADP]/∂x ² + k₁[ATP] − k₂[ADP]; ∂[AMP]/∂t = D ∂²[AMP]/∂x ² + k₂[ADP] − k₃.k_i[AMP]; and ∂[Ado]/∂t = D ∂²[Ado]/∂x ² + k₃.k_i[AMP]. Where D is the diffusion coefficient, and k₁, k₂ and k₃ are rates based on the Michaelis–Menten kinetics given above. These equations were solved numerically using code written for Matlab. We thank Dr. Magnus Richardson for assisting us with this code