|
Figure 1. Anterior endoderm is sufficient to induce cardiogenesis in animal cap cells.Gene expression in explants were analyzed either by RT-PCR (A,B,E) or by in situ hybridization (C). (A) AC/AE conjugates express Nkx2.5 at st. 16, but animal caps and anterior endoderm alone do not. Cardiomyocyte-specific markers MLC2, MHCα and cardiac TnI, as well as cardiogenic transcription factor Tbx5 are all expressed in AC/AE conjugates at st. 34. (B) Posterior Endoderm (PE) is incapable of inducing cardiogenesis in animal caps. (C) Cardiac tissue (MLC2 expression; C1) is induced by anterior endoderm in animal cap·. Animal caps injected with biotinylated dextran were used to make conjugates with anterior endoderm, and after in situ hybridization for cardiac TnI (C2), lineage tracer was revealed by Fast Red staining on sections (C3,4). (D) A drawing of NF st. 10 embryo describing anterior endoderm explants and their subdivision into anterior (A) and posterior (P) parts. Source of drawing: www.xenbase.org. (E) Anterior half of anterior endoderm explants is greatly enriched in Hex expression (St. 10.5) and is a more efficient inducer of cardiogenesis in animal caps at st. 34. (F) Cardiac-inducing activity resides in the anterior (Ant) part of anterior endoderm explants. Double anterior endoderm/animal cap conjugates show two foci of cardiac TnI expression (F1), whereas double posterior endoderm/animal cap conjugates contain only few positive cells (F2). Dissociated and reaggregated anterior endoderm (Diss; F3) induces multiple smaller foci of cardiac gene expression in animal cap (in all 5 positive conjugates; n = 12). F4: control conjugates most frequently have a single cluster of cardiomyocytes (8/8 in this experiment, and 88% of all expressing explants overall, n = 74). |