Claudio D Stern 1 Introduction

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The somites are an intriguing invention of vertebrate embryos. They represent the most overtly segmented structures of the body plan, but they give rise to both obviously segmental (e.g., the axial skeleton) as well as not-so-obvi-ously metameric (dermis and skeletal muscle) elements. In addition, they play a key role in controlling several aspects of the organization of the central and peripheral nervous system of the trunk, and appear to participate in several different types of inductive interactions both within themselves and with neighboring tissues like the neural tube, the notochord, the metanephric and lateral plate mesoderm and the ectoderm and endoderm (see refs. 1 and 2 for reviews).

Questions that can be addressed by manipulating somites range from investigations on the mechanisms by which metameric pattern is established, to their influence on the segmental outgrowth and differentiation of precursors of the peripheral nervous system (neural crest cells, motor axon growth cones), to the control of myogenesis, and patterning and the establishment of regional identities of cells that contribute to the dermis, limbs, and axial skeleton. Previous experiments (1,2) have suggested that although many aspects of somite development are controlled by surrounding tissues, many others appear to be remarkably autonomous.

Somites form at the posterior end of the embryo, such that a pair of somites is added every 100 min or so (see refs. 1 and 2 for reviews). Therefore at any particular stage of development, the embryo contains younger (more recently formed) somites at their caudal end, and older (at more advanced stages following their formation) more rostrally. To indicate this, Ordahl and his colleagues have introduced a "somite stage" numbering system, using Roman numerals to indicate the position of the somite being referred to with respect to

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

its neighbors (see ref. 2). In this system, somites are counted upwards from the most recently formed one, which is designated as I. The most caudal 4-6 somites are usually epithelial spheres. Stages V/VI and higher designate somites whose dorsolateral surfaces still remain epithelial (the dermomyotome; Fig. 1) but whose ventromedial parts have become mesenchymal once more, to form the sclerotome (1,2). The neural tube, notochord, ectoderm and endo-derm all play a role in determining the dorsoventral polarity of the somites with respect to their ability to form a dermomyotome and a sclerotome.

Despite the simplicity of this numbering system, it is important to remember when investigating somite development that overlapped with this age-structure is also a position-dependent address (reviewed in refs. 1 and 2). This can be demonstrated by transplanting somitic mesoderm from the thoracic level to the neck, where they go on to develop ribs as if they had not been transplanted. However, if a similar experiment is conducted to investigate the nature of the muscles that develop, it is found that any somite will give rise to muscles appropriate for its new position. Thus, some somitic derivatives behave in a cell autonomous way concerning their positional information, while others are subject to cues from their environment. In addition, the most rostral 5 or so somites ("occipital somites") have a different fate from the rest (they do not contribute to the vertebral column, some of their cells appear to contribute to the tongue, and in addition, they do not support the development of dorsal root ganglia from neural crest cells migrating within them).

Experiments in which the somitic mesoderm is manipulated can be done either in ovo or in whole embryo (3-5) culture. The main advantage of the former method is that it allows embryos to develop for a long time, even up to hatching, but embryos younger than about the 4 somite stage (see ref. 4 [HH] stage 8) are very difficult to manipulate in this way and their survival is poor. The latter technique allows very young embryos (even before incubation, at preprimitive streak stages) to be operated, but they will only survive for 36-48 h, even in the most expert of hands. In the following sections, I describe two examples. The first is a detailed method for operations on somitic mesoderm in ovo, in which the anterior half of the segmental plate (unsegmented paraxial mesoderm; see Fig. 1) of a quail embryo is grafted into the same position of a host chick embryo. The same procedure is "generic" and can be adapted easily for manipulation of newly formed or older somites at stages 9-15, as well as for manipulations of the notochord, neural tube and other tissues at these stages. The second example, to be used in conjunction with the instructions in the chapter on grafting Hensen's node (see Chapter 16), gives advice on manipulating younger embryos to investigate the mechanism of segmentation from the primitive streak stage onward (see refs. 7 and 8). The procedures given here have been adapted from those in ref. 9.

Fig. 1. Schematic diagrams of embryos at about stage 11. The three regions of the somitic mesoderm can be seen along the axis of the embryo in the upper diagram: unsegmented mesoderm toward the bottom of the drawing, followed by epithelial somites (somite stages I through V or VI), followed more anteriorly by somites that have already split into dermomyotome and sclerotome (somite stages V/VI and higher). Although only three epithelial somites are shown, there are usually five or six of this type in embryos at this stage. The lower diagram represents a transverse section at the level of one of the epithelial somites.

Fig. 1. Schematic diagrams of embryos at about stage 11. The three regions of the somitic mesoderm can be seen along the axis of the embryo in the upper diagram: unsegmented mesoderm toward the bottom of the drawing, followed by epithelial somites (somite stages I through V or VI), followed more anteriorly by somites that have already split into dermomyotome and sclerotome (somite stages V/VI and higher). Although only three epithelial somites are shown, there are usually five or six of this type in embryos at this stage. The lower diagram represents a transverse section at the level of one of the epithelial somites.

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