The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed.

Triclad flatworms are well studied for their regenerative properties, yet little is known about their embryonic development. We here describe the embryonic development of the triclaty 120d Schmidtea polychroa, using histological and immunocytochemical analysis of whole-mount preparations and sections. During early cleavage (stage 1), yolk cells fuse and enclose the zygote into a syncytium. The zygote divides into blastomeres that dissociate and migrate into the syncytium. During stage 2, a subset of blastomeres differentiate into a transient embryonic epidermis that surrounds the yolk syncytium, and an embryonic pharynx. Other blastomeres divide as a scattered population of cells in the syncytium. During stage 3, the embryonic pharynx imbibes external yolk cells and a gastric cavity is formed in the center of the syncytium. The syncytial yolk and the blastomeres contained within it are compressed into a thin peripheral rind. From a location close to the embryonic pharynx, which defines the posterior pole, bilaterally symmetric ventral nerve cord pioneers extend forward. Stage 4 is characterized by massive proliferation of embryonic cells. Large yolk-filled cells lining the syncytium form the gastrodermis. During stage 5 the external syncytial yolk mantle is resorbed and the embryonic cells contained within differentiate into an irregular scaffold of muscle and nerve cells. Epidermal cells differentiate and replace the transient embryonic epidermis. Through stages 6-8, the embryo adopts its worm-like shape, and loosely scattered populations of differentiating cells consolidate into structurally defined organs. Our analysis reveals a picture of S. polychroa embryogenesis that resembles the morphogenetic events underlying regeneration.