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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
3
1
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
3
4
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
5
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
α
-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 (
α
1(II) procollagen gene, Chapter 44), as well as
transcription factors (Msx2, Chapter 44).
Ectopic Expression in Drosophila
9
2
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
Edited by: R. S. Tuan and C. W. Lo © Humana Press Inc., Totowa, NJ
9
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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