developmental biology protocols - Rocky S. Tuan and Cecilia W. Lo.pdf - Developmental biology - annam101

Methods in Molecular Biology TM

VOLUME 137

Developmental

Biology

Protocols

Volume III

Edited by

Rocky S. Tuan

Cecilia W. Lo

HUMANA PRESS

Methods in Molecular Biology TM

Developmental

Biology

Protocols

Volume III

Edited by

Rocky S. Tuan

Cecilia W. Lo

HUMANA PRESS

Overview

Developmental Biology Protocols

Overview III

Rocky S. Tuan and Cecilia W. Lo

1. Introduction

The marriage of cell and molecular biology with embryology has produced remark-

able advances for the field of developmental biology. In this third volume of Develop-

mental Biology Protocols , contemporary, practical methods are first presented for the

analysis and manipulation of developmental gene expression. To illustrate how such

techniques, as well as procedures of experimental embryology including those

described in the first two volumes of the series, may be applied in the study of develop-

ment, a panoramic collection of experimental models of morphogenesis, development,

and cellular differentiation are detailed. Both in vivo and in vitro systems are included.

The volume concludes with various examples of developmental models of diseases

and their molecular basis.

2. Manipulation of Developmental Gene Expression and Function

Drosophila has been and remains one of the most versatile model systems for the

manipulation of developmental gene expression. Chapter 2 focuses on a description of

the experimental approaches currently used in ectopic gene expression in Drosophila

to examine the function of a given gene in the desired tissue. Chapter 3 deals with the

utilization of the highly efficient FLP/FRT yeast site-specific recombination system to

generate somatic and germline clones in Drosophila for phenotypic analysis and screening.

Chapters 3 and 4 address the methods used to alter gene expression as well as gene

function in another experimentally highly accessible system, the developing chick

embryo. Chapter 3 describes the application of antisense oligonucleotides to “knock

down” gene expression in somitic stage chick embryos, whereas Chapter 4 discusses

how functional neutralizing monoclonal antibodies may be used to block the activity of

a specific gene product, N-cadherin, in the developing chick embryonic limb bud.

3. Analysis of Gene Expression

The first step in analyzing the molecular basis of any developmental event is to

characterize and compare gene expression profiles, both spatial and temporal, as a function

From: Methods in Molecular Biology, Vol. 137: Developmental Biology Protocols, Vol. III

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

Tuan and Lo

of development. A comprehensive list is provided in this section. Classic methods such

as Northern blotting is not presented here, because relevant protocols are readily avail-

able in many technical manuals of molecular biology. Quantitative methods include

ribonuclease protection assay (Chapter 6), and polymerase chain reaction (PCR) based

methods (Chapters 7 and 8). In situ hybridization (Chapters 9–15) has gained wide

application in visualizing the spatial aspects of gene expression in the developing

embryo, particularly in mapping the dynamics of tissue morphogenesis. In particular,

the ability to carry out multiple in situ hybridizations (Chapter 14), or sequential in situ

hybridization and immunohistochemistry (Chapters 12 and 15), on a given specimen

should be invaluable for analyzing the potential roles of genes and gene products in

development.

The potential of the green fluorescent protein (GFP) of the jellyfish, Aequoria

victoria , as a vital recombinant tag for genes of interest has produced a great deal of

excitement in developmental biology; Chapter 16 provides a thorough discussion of

the principles and techniques in the application of the GFP. Finally, the basic strategy

in the application of monoclonal antibodies, one of the most powerful technical

advances in modern biomedical research that has enjoyed a distinguished history, in

the study of embryonic development is presented (Chapter 17).

4. Models of Morphogenesis and Development

This section presents a number of developmental model systems under active inves-

tigation to illustrate the multitude of experimental questions currently being addressed

in the field of developmental biology. The inductive events of embryogenesis and

means for their analyses are described in Chapters 18 and 19. Techniques for whole or

partial embryo explant cultures for the somitic stage embryos for the analysis of meso-

dermal and neural crest studies are covered in Chapters 20 and 21. Other models of

morphogenesis include those for angiogenesis (Chapter 22), vasculogenesis (Chapter 23),

and epithelial–mesenchyme interactions (Chapter 24). Specific organogenesis models

are also included—limb bud (Chapter 25) and palate (Chapter 26).

5. In Vitro Models and Analysis of Differentiation and Development

Regulation of cell differentiation is one of most active research areas of develop-

mental biology. With the advent of cell and molecular biology, and the identification of

differentiation-associated genes, cell differentiation is often interpreted in terms of

regulation of gene expression. Both cis and trans modes of gene expression regulation

have been found to operate during cell differentiation, leading to active investigation

on structure/function of gene promoters and transcription factors.

This section is a collection of many in vitro cell differentiation systems currently

under active investigation. Early events in development include fertilization (Chapter 27)

and trophoblastic differentiation (Chapter 28). Bone marrow-derived mesechymal pro-

genitor cells have received a great deal of recent attention as candidate cells for cell-

based tissue engineering. It is generally believed that the differentiation potentials of

these cells represent a partial recapitulation of the characteristics of embryonic meso-

dermal cells. Techniques for their isolation, culture, and characterization are described

in Chapter 29. Another cell type important for studying cell differentiation are germ

cells; methods for their isolation and culture are included in Chapter 30. Prostate cell

Overview

differentiation is discussed in Chapter 30. Cell differentiation in connective tissues is

presented in the following chapters: striated muscle differentiation (Chapter 31),

somitic myogenesis (Chapter 32), mesenchymal chondrogenesis (Chapters 33–35), and

bone cell differentiation (Chapter 36).

In addition to specific examples and systems of cellular differentiation, methods for

three crucial aspects of cellular activities are also presented. Cell–cell interaction is

illustrated in Chapter 39, which deals with cadherin-mediated events. Cell–matrix inter-

actions as mediated by hyaluronan binding are discussed in Chapter 40. The dynamic

regulation of cytoskeletal architecture, visualized and analyzed by the microinjection

of fluorescently-labeled

-actinin into living cells, is presented in Chapter 41.

6. Developmental Models of Diseases

The experimental paradigms gained from developmental biology lend readily to the

mechanistic analysis of diseases. Several examples are included here. Pax 3 , a member

of the vertebrate Pax gene family containing a DNA-binding domain known as the

paired domain, is important for proper formation of the nervous, cardiovascular, and

muscular systems. The molecular analysis of Pax 3 mutations and how the pathways

affected lead to the pathogenesis of specific dysmorphogenic consequences is the sub-

ject of Chapter 42. Finally, one of the most powerful contributions of molecular devel-

opmental biology to the study of diseases is the application of transgenic methodologies

to create animal models of human diseases. The three examples included here all deal

with various aspects of skeletal defects, including both trunk as well as craniofacial

malformations. The methods involve studies utilizing a structural gene (collagen type X,

Chapter 43), cell specific promoter (

1(II) procollagen gene, Chapter 44), as well as

transcription factors (Msx2, Chapter 44).

Ectopic Expression in Drosophila

Elizabeth L. Wilder

1. Introduction

Ectopic expression in Drosophila has been used extensively to examine the capa-

bilities of a given gene in virtually any tissue. Three general approaches are described

here, and the choice of which to use is determined by the needs of the particular experi-

ment. Certain aspects of each approach can also be combined, providing powerful tools

for the examination of gene function. Because ectopic expression does not involve a

protocol, but rather generation of certain types of transgenic strains, this chapter focuses

on a description of the approaches and in what circumstances each is likely to be useful.

2. Materials

For each of the methods of ectopic expression described here, the production of

transgenic strains is required. The vectors that are widely used in these experiments are

available (1–3) .

3. Methods

3.1. Expression Through Defined Promoters

The simplest means of ectopic expression is through the construction of a promoter-

cDNA fusion in which a gene of interest is driven by a defined promoter or enhancer.

Transgenic strains carrying this construct then ectopically express the gene of interest

in the defined pattern.

One of the most commonly used promoters for this purpose is the heat shock protein

70 (hsp70) promoter (1) . This promoter allows ubiquitous expression to be induced in

any tissue of the fly through a simple heat shock at 37

From: Methods in Molecular Biology, Vol. 137: Developmental Biology Protocols, Vol. III

C. The inducible nature of

this approach is a great advantage. However, basal levels of expression can be prob-

lematic, and heat shock itself can induce developmental defects. In addition, short

bursts of ectopic expression ubiquitously is often not ideal. Therefore, sustained

expression in defined domains may be preferred.

To achieve ectopic expression within a defined domain, transcriptional regulatory

regions from characterized genes have been linked to genes of interest (4 , 5) . The advan-

tage of this approach is its simplicity. Its primary limitation is that lethality can result

from the ectopic expression. This makes it impossible to establish stable transgenic

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