Developmental Biology Protocols [Vol 1] [Methods in Molec Bio 135] - R. Tuan, C. Lo (Humana) WW.pdf - książki - Bio Med 08 - bibiariel

Methods in Molecular Biology TM

VOLUME 135

Developmental

Biology

Protocols

Volume I

Edited by

Rocky S. Tuan

Cecilia W. Lo

HUMANA PRESS

Developmental

Biology

Protocols

Volume I

Edited by

Rocky S. Tuan

Cecilia W. Lo

Overview

Developmental Biology Protocols

Overview I

Rocky S. Tuan and Cecilia W. Lo

1. Introduction

As the next millennium dawns, developmental biology, the study of the processes

that give rise to cellular diversity and order within an organism and to the continuation

from one generation to the next, has reached a most exciting stage as an experimental

science. In particular, in the last two decades, the application of analytical and techni-

cal know-hows generated from the “molecular biology revolution” have critically

advanced our understanding of development in a mechanistic way. There is every rea-

son to believe that the study of development will be one of the most promising areas of

the life sciences in the next millennium.

The goal of this three-volume set of Developmental Biology Protocols is to provide

the reader with a richly annotated compendium of protocols representing current, state-

of-the-art experimental approaches used in the study of development. The scope of the

volumes is intentionally broad, as modern developmental biology is by necessity a

wide-ranging discipline, involving multiple experimental systems, as well as using

techniques generated from many fields. This chapter provides a brief overview of the

protocols covered in this volume.

2. Systems: Production, Culture, and Storage

Beginning with Aristotle’s elegant descriptive treatise on avian embryonic develop-

ment (doubtlessly prompted by the incorporation of eggs as a food staple!), the use of

animal model systems has been one of the most important aspects of the study of devel-

opment. This volume has selected three model systems, echinoderm (sea urchin; Chap-

ters 2 and 3), avian (chicken; Chapters 4, 5, and 6), and rodents (mouse; Chapters 7, 8,

and 9), to illustrate the requirements and rationales for using particular model systems

for the study of embryonic development. Readers are advised to consult other more

specialized literature sources exclusively dedicated to a particular system for similar

information on other experimental model systems of development, such as Xenopus ,

Coenorhabditis elegans , Drosophila , and zebrafish.

From: Methods in Molecular Biology, Vol. 135: Developmental Biology Protocols, Vol. I

Edited by: R. S. Tuan and C. W. Lo © Humana Press Inc., Totowa, NJ

Tuan and Lo

3. Developmental Pattern and Morphogenesis

This section focuses on how the pattern and formation of specific organs and tissues

may be experimentally examined. Examples include the analysis of inductive interac-

tions (Chapter 11) and gastrulation and mesodermal patterning (Chapter 12), and the

examination of head and brain (Chapter 10), craniofacial (Chapter 13) and axial skel-

etal development (Chapter 14), as well as cardiac morphogenesis (Chapter 15).

4. Embryo Structure and Function

The study of embryonic development depends on the precise analysis of structure

and function in order to detect changes in form and shape as well as biological activi-

ties, particularly if experimental perturbations are performed. This section provides

state-of-the-art methodologies for histological and immunohistochemical analyses

(Chapters 16, 18, and 20), and high-resolution imaging using confocal laser scanning

microscopy (Chapters 17, 19, and 20) and ultrasound backscatter microscopy (Chapter 23).

Functional analyses include magnetic resonance imaging (Chapter 21), optical coher-

ence tomography (Chapter 22), Doppler echocardiography (Chapter 24), and cellular

calcium imaging (Chapter 25). The exciting application of information technology to

imaging is highlighted in Chapter 26, which describes softwares developed for the

acquisition, display and analysis of digital three-dimensional time-lapse data sets.

5. Cell Lineage Analysis

One of the ongoing challenges of developmental biology is to map the origin

and the fate of progenitor cells in the course of tissue patterning and morphogen-

esis. This section presents examples of the many markers and microscopic imaging

methods currently used. Cell labeling with fluorescent dyes is described (Chapters 30,

33, and 34). Gene markers, introduced recombinantly into specific cell populations, are

powerful tools for cell lineage analysis (Chapters 27, 28, and 29). These approaches,

coupled with new microscopic and digital computing instrumentations (e.g., Chapter 31),

have provided exciting new information on cell lineage during development in many

model systems.

6. Chimeras

Chimeras refer to individuals made up of the parts of more than one individual.

Experimentally, by grafting cells or tissue from one embryo (donor) to another (host),

transplantation chimeras can be produced in many species and often between species.

Provided specific detection methods are available, such chimeras allow the investiga-

tor to follow a specific group of cells (the graft) through a period of development and to

determine the fates and locations of their progeny. Chapters in this section cover mul-

tiple systems and approaches in using the chimera technology, both intra- and interspe-

cific. Because of the oviparous nature of their development, avian embryos, specifically

those of the chicken and quail, have long been used to generate transplantation chime-

ras (Chapters 35, 36, and 37). Recently, grafting technology has also been developed

for mouse embryos (Chapter 39), as well as for interspecific chimeras, particularly in

the analysis of neural crest cells (Chapter 40) and somites and neural tube (Chapter 41).

For mouse embryos, the establishment of the embryonic stem cell (ES) technology has

Overview

been one of the most important advances in transgenesis. The utilization of ES cells in

the production of chimeras to permit developmental analysis is covered in Chapter 38.

In the case of C. elegans , an animal whose nearly invariant cell lineage has been fully

described, the use of genetic mosaics (i.e., individuals that harbor both genotypically

mutant and genotypically wild-type cells), has been invaluable in determining the cells

that need to inherit a functional copy of a gene in order to prevent a mutant phenotype

(Chapter 42).

7. Experimental Manipulation of Embryos

A common theme in most of the chapters of this volume is the versatility of the

developing embryo as an experimentally accessible system. In fact, it is the prospect of

applying contemporary analytical tools to revisit “experimental embryology” that is

creating the excitement among modern developmental biologists. This section describes

some of the current methodologies in experimental embryology: (1) carrier-mediated

delivery of growth factors (Chapter 43); (2) laser ablation and fate mapping (Chapter 44);

(3) photoablation of cells expressing

-galactosidase is

of great potential application in assessing the functional importance of specific cell

populations in development.

8. Application of Viral Vectors in the Analysis of Development

Retrovirus and adenovirus are the two most commonly used viral vectors for gene

transduction in vertebrates. This section details the protocols in the construction and

production of retroviral vectors (Chapter 47), the application of retroviral vectors in

gene transduction in limb mesenchyme cultures (Chapter 48), and the construction of

adenoviral vector (Chapter 49) and its application in the analysis of eye development

and cardiovascular development (Chapters 50 and 51).

Volume I provides the reader with sophisticated and current information on issues

of primary importance to experimental developmental biology. Practical details on the

acquisition and setting up of the appropriate experimental model system, the means to

analyze embryonic structure/function, the ways to perturb these processes both experi-

mentally as well as taking advantage of current recombinant techniques, and the analy-

sis of cell lineage, should all be of great utility to both the beginning and seasoned

developmental biologists.

-galactosidase (Chapter 45); and (4) ex utero

surgery (Chapter 46). Given that many transgenic animals used in the study of devel-

opment harbor the LacZ reporter gene under the regulation of promoters of putative

importance, the ability to specifically ablate those cells that express

Rearing Larvae

Rearing Larvae of Sea Urchins

and Sea Stars for Developmental Studies

Christopher J. Lowe and Gregory A. Wray

1. Introduction

Sea urchins have long been used to study morphogenesis and cell fate specification

and are an established model system in developmental biology (1) . Most contemporary

studies have focused on early development, however, and few molecular genetic

studies have examined larval development, or the formation of the highly derived

radial body plan of the adult (2) . A better understanding of the molecular genetic basis

of both the body plans of this phylum may contribute significantly to several fields of

biology (3 , 4) .

Despite over a century of debate, the evolution of the chordate body plan from its

invertebrate ancestors is still a contentious issue (5–8) . As a group closely related to

the chordates (8) , echinoderms are in a crucial phylogenetic position for reconstructing

the evolution of the chordate body plan (7) . The common ancestor of hemichordates,

echinoderms, and chordates may have had a larva that resembled the early feeding

larva of echinoderms (5) . Garstang proposed that the ciliated band of such a larva was

modified by a dorsal fusion, resulting in the formation of structure that was further

modified to become the chordate neural tube. A greater understanding of the molecular

genetics of echinoderm larval development may provide critical insights into the evo-

lution of key chordate innovations such as the neural tube and notochord (8) .

The orthologs of many body-patterning genes present throughout the bilateria have

been isolated from echinoderms (9 , 10) . Understanding how these genes (seemingly so

conserved in patterning the embryos of diverse metazoans), function to establish the

echinoderm radial adult body secondarily from a bilateral larva should provide insights

into the role of animal body-patterning genes in morphological evolution (3 , 4) . Pre-

liminary studies have proposed that the evolution of many novel aspects of echinoderm

morphology was associated with recruitment of body-patterning genes into several new

developmental roles (2 , 11) .

Larval culturing techniques are described for three echinoids ( Lytechinus variegatus ,

Strongylocentrotus purpuratus , and Strongylocentrotus droebachiensis ) and one aster-

oid ( Pisaster ochraceus ). These species were chosen based primarily on practical con-

siderations, adult availability and robustness, length of reproductive season, and ease

From: Methods in Molecular Biology, Vol. 135: Developmental Biology Protocols, Vol. I

Developmental Biology Protocols [Vol 1] [Methods in Molec Bio 135] - R. Tuan, C. Lo (Humana) WW.pdf

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