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The adult Eastern newt, Notophthalmus viridescens, has long served as a model for appendage as well as heart muscle regeneration studies. Newt tissues include all major cell types known in other vertebrates and mammals, including bone, cartilage, tendon, muscle, nerves, dermis, and epidermis. Therefore, these aquatic salamanders make an excellent model for studying the regeneration of complex tissues. Regeneration of adult tissues requires the integration of new tissues with preexisting tissues to form a functioning unit through a process that is not yet well understood. Scale is also an issue, because the regenerating tissues or structures are magnitudes larger than their embryonic counterparts during development, and therefore, it is likely that different physics and mechanics apply. Regardless, regeneration recapitulates to some degree developmental processes. In this chapter, we will describe basic methods for maintaining adult Eastern newts in the laboratory for the study of regeneration. To determine similarities and differences between development and regeneration at the cellular and molecular level, there is also a need for embryonic newt tissue. We therefore also outline a relatively simple way to produce and raise newt embryos in the laboratory.

For more than 2,000 years, philosophers and scientists have turned to the avian embryo with questions of how life begins (Aristotle and Peck Generations of Animals. Loeb Classics, vol. XIII. Harvard University Press, Cambridge, 1943; Needham, A history of embryology. Abelard-Schuman, New York, 1959). Then, as now, the unique accessibility of the embryo both in terms of acquisition of eggs from domesticated fowl and ease at which the embryo can be visualized by simply opening the shell has made avians an appealing and powerful model system for the study of development. Thus, as the field of embryology has evolved through observational, comparative, and experimental embryology into its current iteration as the cellular and molecular biology of development, avians have remained a useful and practical system of study.

The use of magnetic resonance imaging of the fetal rat and rabbit head, as an alternative to the traditional methods of fixation and preparation of serial sections, is described. Labeled magnetic resonance images of normal head anatomy have been provided as a reference for use when evaluating the internal structures of the head.

A skeletal examination of fetuses is required in regulatory embryo-fetal development studies. This chapter describes a method of skeletal examination using alizarin staining. All fetuses are removed from the mother by caesarean section before birth. The fetuses are first examined externally. For larger species (rabbit and minipig), an internal examination can be performed on the fresh soft tissues by microdissection. For smaller species, such as the rat and mouse, half of each litter is fixed for internal soft tissue examination. The other half is used for skeletal examination. A rapid examination of the fresh soft tissues is performed before evisceration and fixation. The staining process takes several days. Following staining, all bones are examined from the head to the tail, in ventral and dorsal positions.

Embryo fetal development (EFD) studies in nonhuman primates are frequently conducted in macaques with Macaca fascicularis (cynomolgus monkey, long-tailed macaque, crab-eating macaque) being the most accepted model. EFD studies are also feasible in the marmoset. Due to recent guideline changes (ICH M3(R2) and S6(R1)), EFD studies are largely confined to conventional pharmaceutical compounds rather than biopharmaceuticals. This chapter describes basic study designs and provides working protocols for collecting, processing, and staining fetuses, including collection of amniotic fluid and umbilical cord blood. The chapter also covers the examination and terminology for external, visceral, and skeletal examinations of fetuses. The species covered in this chapter are cynomolgus monkey (long-tailed macaque) and marmoset monkey.

This chapter describes methods for the examination of fetal abdominal and thoracic soft tissues by microdissection on either fresh (non-rodent) or fixed (rodent) specimens in order to detect structural abnormalities. With hundreds of fetuses examined for each species (rodent and non-rodent) in regulatory reproductive toxicity assessments (ICH, http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf, 2009; ICH, http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S5_R2/Step4/S5_R2__Guideline.pdf, 2005), microdissection techniques allow a thorough and relatively rapid examination of fetuses for soft tissue abnormalities.

The assessment of developmental toxicology data is a critical aspect of hazard evaluation for pharmaceuticals and environmental chemicals. Skeletal examination is an essential part of prenatal developmental toxicity studies of chemicals as well as pesticides and comprises evaluation of both cartilaginous and ossified skeletal components. Various techniques are published in the literature to process and double-stain skeletons of common laboratory animals which are all based on staining of the cartilage with Alcian Blue and staining of ossified bones with Alizarin Red S along with maceration (clearing) of the surrounding soft tissue. The staining of the cartilage allows the examination to assess ossified structures and their underlying cartilage in a single step.

Micro-computed tomography imaging technology allows for the whole-mount investigation of skeletal structures in preclinical specimens at both fetal and postnatal time points. The imaging process is nondestructive to the specimen and can be performed at various resolutions to derive the region of interest information most relevant to individual researchers. In addition to qualitative imaging of skeletal samples, accurate and desirable metrics such as bone mineral density (BMD), discrete cortical and trabecular bone analysis, and milligrams hydroxyapatite per unit volume (mgHA/cc) are also achievable depending upon scanner platform. Additional benefits to the method include digitally archivable files, in vivo and ex vivo scanning options, and volumetric or slice-thru presentation of data in standard histological or oblique orientations. Some drawbacks to the method include long scan times at higher spatial resolutions, large file sizes, and limitation to imaging of highly dense biological structures (i.e., bone), though several groups have attempted to expand the modality to include soft tissue imaging in ex vivo specimens.

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

Reptiles (lizards, snakes, turtles and crocodylians) are becoming increasing popular as models for developmental investigations. In this review the leopard gecko, Eublepharis macularius, is presented as a reptilian model for embryonic studies. We provide details of husbandry, breeding and modifications to two popular histological techniques (whole-mount histochemistry and immunohistochemistry). In addition, we provide a summary of basic reptilian husbandry requirements and discuss important details of embryonic nutrition, egg anatomy and sex determination.

The ability to combine embryological manipulations with gene function analysis makes the chick a valuable system for the vertebrate developmental biologist. We describe methods for those unfamiliar with the chick wishing to initiate chick experiments in their lab. After outlining how to prepare chick embryos, we provide protocols for introducing beads or cells expressing secreted factors into the embryo and for culturing tissue explants as a means of assessing development in vitro. Chick gain-of-function and loss-of-function (RNAi and morpholino oligonucleotide) approaches are outlined, and methods for introducing these reagents by electroporation are detailed.

Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.

Cell movements occur in all phases of animal life from embryogenesis, to maintaining adult organs, to comprising a critical component of pathology. During gastrulation, cells demonstrate a repertoire of morphogenetic movements coordinated with fate inductions to sculpt the embryonic body. The morphogenetic behaviors, underlying mechanisms, and their control, are the subject of much current study. External development of the transparent zebrafish embryo, the abundance of mutations influencing cell movements, as well as a range of observation and manipulation methods, make the zebrafish valuable for cell movement studies. This chapter offers a conceptual background for analysis of gastrulation cell movements by reviewing how region specific cell movements shape the wild-type zebrafish embryo, and how defective morphogenetic movements alone or in combination with altered cell fate specification distort the body plans of known zebrafish mutants. We furnish methods for the morphometric analysis of embryonic shape and organ rudiments in live and fixed embryos, and present data collected from live wild-type, dorsoventral patterning (somitabun and chordino) and convergence and extension (knypek and trilobite) classes of mutants. We provide a method for quantitative assessment of the movements of cell populations in vivo, and a method for determining whether cell fate and/or movement are disturbed.