Fig. 1. Animal cap assay and sandwich method as in vitro induction systems. In amphibians, a blastocoel cavity clearly forms inside the animal hemisphere during the blastula and early gastrula stages. The cap-like portion lining the roof of the blastocoel cavity is the animal cap. This region consists of a sheet of pluripotent cells, organized into one or several layers. In the animal cap assay, the animal cap was treated with a physiological saline solution containing inducing factors and then cultured. Depending on the type, concentration, and duration of exposure to the inducing factors, animal caps can differentiate into various cell types. In contrast, the sandwich method, involves culturing the inducer source in between two animal caps. In this technique, the sources of induction can include the dorsal lip of the blastopore (organizer), adult tissues, pelletized soluble factors, or animal caps pretreated with soluble factors. In this figure, activin is used as an example of an inducing factor.
![Fig. 12. Summary of the in vitro induction system using activin as an inducing factor.
This in vitro induction system utilizes activin and retinoic acid as inducing factors to treat animal caps, employing techniques such as animal cap assay, dissociation/reaggregation protocol, and the sandwich method. By applying these methods, various levels of self-organization can be replicated and controlled in vitro, ranging from lower-order cell differentiation to higher-order tissue differentiation, organogenesis, and even the formation of fundamental body plans. Abbreviations: Dorsal [D], ventral [V], and retinoic acid [RA].](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:cnykzbo2impysrf6a6scy6oh/bafkreiarcsezsnfoj4xmlow6thcxsfy6wsfxfujek53lgt6wsw2k2wshqe)
Fig. 12. Summary of the in vitro induction system using activin as an inducing factor. This in vitro induction system utilizes activin and retinoic acid as inducing factors to treat animal caps, employing techniques such as animal cap assay, dissociation/reaggregation protocol, and the sandwich method. By applying these methods, various levels of self-organization can be replicated and controlled in vitro, ranging from lower-order cell differentiation to higher-order tissue differentiation, organogenesis, and even the formation of fundamental body plans. Abbreviations: Dorsal [D], ventral [V], and retinoic acid [RA].
![Fig. 11. Formation of embryoids by artificial activin concentration gradients.
To create embryoids, animal caps were prepared through treatment with low (0.5–1 ng/ml), intermediate (5–10 ng/ml), or high (50–100 ng/ml) concentrations of activin. These three types of activin-treated animal caps were then sequentially arranged and cultured with untreated animal caps. After 3 days of culture, embryoids with distinct head and trunk-tail structures were formed (A). Histological sections revealed differentiation into head tissues, such as the cement gland [cg] and eyes, and trunk-tail tissues including the ear vesicle [ev], brain [br], notochord [not], muscle [mus], and gut (B). When newt embryos are used in similar combination cultures, neural plate structures forming the brain [white arrow] and axial structures forming the trunk-tail regions [black arrow] are sometimes observed (C).](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:cnykzbo2impysrf6a6scy6oh/bafkreig36tdo3ewakfxhn66uzs2kfqlkjjoebdcfh5rzy3ubxkfnn7t5iu)
Fig. 11. Formation of embryoids by artificial activin concentration gradients. To create embryoids, animal caps were prepared through treatment with low (0.5–1 ng/ml), intermediate (5–10 ng/ml), or high (50–100 ng/ml) concentrations of activin. These three types of activin-treated animal caps were then sequentially arranged and cultured with untreated animal caps. After 3 days of culture, embryoids with distinct head and trunk-tail structures were formed (A). Histological sections revealed differentiation into head tissues, such as the cement gland [cg] and eyes, and trunk-tail tissues including the ear vesicle [ev], brain [br], notochord [not], muscle [mus], and gut (B). When newt embryos are used in similar combination cultures, neural plate structures forming the brain [white arrow] and axial structures forming the trunk-tail regions [black arrow] are sometimes observed (C).
![Fig. 7. In vitro heart formation and in vivo transplantation experiment.
When treated with a high concentration of activin, the animal caps of Xenopus embryos did not differentiate into heart tissue. However, if the animal cap dissociates into individual cells before activin treatment and then reaggregates, it forms a beating heart [arrow] with 100 % efficiency (A). This heart expresses differentiation marker genes, such as Nkx2.5, GATA-4, Tbx5, MHCα, TnIc (cardiac troponin I), and ANF, none of which are expressed in an animal cap treated with activin alone, without dissociation/reaggregation (B). Electron microscopy reveals the presence of intercalated discs [id] specific to the cardiac muscle, along with visible mitochondria [m] and Z-bands [z] (C). When the reaggregated heart tissue is orthotopically transplanted into the cardiac primordium of a neurula-stage embryo, it integrates without rejection and continues to beat (D), although it does not persist through host metamorphosis. In contrast, when the reaggregated tissue is ectopically transplanted into the ventral region of the neurula, it begins to beat synchronously with the host heart and gradually reddens as it initiates blood circulation (E).](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:cnykzbo2impysrf6a6scy6oh/bafkreig77lwzs67jdcjwi2gjsany3shyoa5vj2dpetburc3pegwxeb45a4)
Fig. 7. In vitro heart formation and in vivo transplantation experiment. When treated with a high concentration of activin, the animal caps of Xenopus embryos did not differentiate into heart tissue. However, if the animal cap dissociates into individual cells before activin treatment and then reaggregates, it forms a beating heart [arrow] with 100 % efficiency (A). This heart expresses differentiation marker genes, such as Nkx2.5, GATA-4, Tbx5, MHCα, TnIc (cardiac troponin I), and ANF, none of which are expressed in an animal cap treated with activin alone, without dissociation/reaggregation (B). Electron microscopy reveals the presence of intercalated discs [id] specific to the cardiac muscle, along with visible mitochondria [m] and Z-bands [z] (C). When the reaggregated heart tissue is orthotopically transplanted into the cardiac primordium of a neurula-stage embryo, it integrates without rejection and continues to beat (D), although it does not persist through host metamorphosis. In contrast, when the reaggregated tissue is ectopically transplanted into the ventral region of the neurula, it begins to beat synchronously with the host heart and gradually reddens as it initiates blood circulation (E).
A fascinating review on the role of Activin in organ induction. Isn't it wild that in Xenopus embryos, a piece of the animal cap can be induced with Activin at different concentrations and buffers to form the ❤️, kidney, the pancreas, head, tail, and even a whole embryoid 🤯:
doi.org/10.1016/j.cd...
