Cultured embryonic and adult skeletal muscle cells have a number of different uses. The microdissected explant technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or em ...

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages, requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive ...

The gastrointestinal tract of vertebrates is lined by epithelium that develops from the endodermal germ layer. The oesophagus and intestine form part of the gastrointestinal tract and studying the normal development of both tissues is difficult due to lack of suitable in vitro model syst ...

Culturing embryonic tissue in an in vitro setting offers the unique ability to manipulate the external medium and therefore to investigate the pathways involved in regulating normal organogenesis as well as providing models for developmental disorders. Here we describe a system for t ...

Muscle tissue culture provides a system for studying the growth and differentiation of muscle cells in a controlled environment. In mature muscle tissue, terminally differentiated myocytes form multinucleate syncytia in which structural and regulatory genes are expressed and t ...

Detailed cytological analysis of cells undergoing differentiation often reveals clues to the regulation of multiple cell features. The Hedgehog (Hh) signaling cascade is a master regulator of cell fate during differentiation and is implicated in the development of some neoplasia ...

We describe a method of isolating and maintaining primary cultures of mouse neonatal cardiac myocytes (NCM). This is derived from the well-established procedure for making NCM cultures from rat neonates by sequential digestion of rat ventricular myocardial pieces using a collagena ...

Efficient manipulation of Hedgehog (HH)/GLI signaling activity is crucial to the analysis of molecular events underlying HH/GLI-regulated cell fate determination and tumor growth. In this article, we describe the use of retroviral expression systems as a valuable tool to activate or r ...

Over the recent years, a large number of embryological studies with the zebrafish have provided substantial evidence of its usefulness for the investigation of the genetic and cellular basis of vertebrate development. With regard to the Hedgehog (Hh) pathway, forward as well as reverse gen ...

Multiple assays for Shh activity using cell lines, primary cultures, and explanted tissue have been described. We first described the use of E11.5 rat dorsal telencephalic explants to assay Shh ventralizing and differentiation-inducing activity in Kohtz et al. (1). Using this assay, we sub ...

Xenopus embryos provide a powerful model system to investigate the complex molecular mechanisms, which are controlled by or control the activity of the Hedgehog (Hh) signaling pathway. The use of synthetic mRNA or antisense oligonucleotide (morpholino) microinjection into blast ...

Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polari ...

The purification of recombinant versions of the N-terminal signaling fragment of Sonic hedgehog (ShhN) from E. coli, Hi-5™ insect cells, yeast, and mammalian cell sources reveals diverse post-translational modifications that affect the potency of the purified protein. Modificati ...

Sequence analysis and comparative genomics are powerful tools to gain knowledge on multiple aspects of gene and protein regulation and function. These have been widely used to understand the evolutionary history and the biochemistry of Hedgehog (Hh) proteins, and the molecular contr ...

The Hedgehog (Hh)-signaling pathway has been intensively studied in the past decade. Increasing evidence suggests that dynamic formation of protein complexes plays a critical role in the organization and regulation of Hh signaling. Immunoprecipitation (IP) is a powerful tool to stu ...

This chapter describes how to perform basic biochemical fractionations of Drosophila cells, and how to begin to characterize the proteins in the resulting fractions. The protocols include maintenance and transfection of Drosophila cell lines (Section 3.1.), hypotonic lysis (Sect ...

The GAL4/upstream activating sequence (UAS) system is one of the most powerful tools for targeted gene expression. It is based on the properties of the yeast GAL4 transcription factor which activates transcription of its target genes by binding to UAS cis-regulatory sites. In Drosophila, the ...

To fully understand how animals develop, it is often necessary to remove the function of a particular gene in a specific cell type or subset of cells. In Drosophila melanogaster, mosaic animals have been widely utilized to study cell fate, growth and patterning, and restriction of cell fate. This cha ...

Many of the genes of Drosophila melanogaster have their transcripts deposited in developing oocytes. These maternally loaded gene products enable an otherwise homozygous mutant embryo to survive beyond the first stage of development for which the gene product is required. Zygotic mu ...

RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene to examine its involvement in biological processes. Many strategies exist to reduce ...