Download finding the genes that regulate development

Developmental Biology BY1101
P. Murphy
Lecture 9
Developmental genetics: finding the genes that regulate development
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Introduction
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The application of genetic analysis and DNA technology to the study of
development has brought about a revolution in our understanding of how a
complex multicellular organism develops from a single cell.
Three advances in particular had a major impact
(1) The isolation and study of genes that regulate development, originally in the
fruitfly Drosophila. These are called Developmental Regulatory Genes; also known
as “Master regulators”
(2) The realisation that such genes and basic developmental events are extremely
highly conserved through evolution. So observations made in one organism (e.g.
Drosophila) have wider relevance to all animals (including humans).
(3) The development of molecular techniques to manipulate developmental genes
and investigate their function in model organisms. For example: (1) Methods to
investigate in which cells in the embryo a particular gene is expressed (“turned on”)
(called in situ hybridisation); (2) Methods to generate transgenic animals, adding
genes to the genome to examine their effect; (3) Methods to specifically remove or
inactivate genes as have been developed for the mouse (“knock-out mice”), allowing
direct analysis of gene function. These allow biologists to begin to understand what
a genes’ normal function is by seeing the effect of removing it.
In lecture 9 we began by looking at the history of how biologists started to examine
the genetics of development and then particularly how geneticists used the fruit
fly to uncover evidence of the first developmental regulatory genes.
Historical perspective: The importance of genetics in guiding development
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The groundwork for our understanding of development was laid by detailed
anatomical observations of embryology. Mostly descriptions of developing
embryos and the effects of moving bits of embryos about. Up until the middle
of the 20th century, development was being studied largely at the descriptive
and anatomical level with the emphasis on amphibian and bird development.
The relevance of genetics to development was not initially appreciated.
Genetics was initially seen as inheritance from generation to generation with little
relevance to cellular differentiation.
The link between genetics and development was not appreciated until the nature
and function of genes was better understood from the 1940s on. That is that genes
code for proteins and the compliment of proteins expressed in a given cell
determines its phenotype or characteristics.
Accessing Developmental genes:
Realising that genes could play an important role in guiding development led to new
interest in trying to find such genes and find out what kind of gene products
(proteins) these genes encode; the big question was how could a gene product guide
a developmental event? To understand development at the molecular genetic level
we had to have access to the genes that govern and guide development in order to
study them and find out how they operate.
But first, how could such genes be identified and isolated?
In this lecture (9) we dealt with how genetic regulators of development in the fruitfly
Drosophila were identified.
In lecture 10 we will see how this has benefited and influenced the study of
development in other organisms.
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Question: How can we find the genes that guide development?
Answer: By studying individual organisms in which these genes have been altered developmental mutants.
Developmental genes have therefore classically been identified in two ways
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From spontaneous mutations arising in laboratory stocks of a model organism.
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Relatively very few.
From large scale mutant screens where animals are exposed to mutagens (chemical
or radiation) to increase the frequency of genetic damage. The animals are then bred
and the offspring screened to see if any alterations to the genes had produced a
change (mutant phenotype) in the characteristic of interest e.g. changing the shape,
size or appearance of a part of the body.
It is not easy to carry out mutational screens but it is easier in some organisms than in
others.
The feasibility of a mutant screen depends on a number of factors:
• Space to house large numbers of animals.
• Person hours to screen large numbers of individuals.
• Phenotypes that are easy to observe.
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Short generation interval to accommodate breeding several generations.
A simple genome that is easy to map and characterise.
The fruitfly Drosophila is particularly well suited to genetic screens of this kind for
all of the above reasons.
In addition these animals have another important advantage for this work:
Fruitflies and other arthropods have a modular construction; an ordered series of
segments in their body plan.
Each segment is anatomically distinct, with characteristic appendages, so that
alterations to the structure are easier to identify.
Through identifying mutations that altered or interfered with this segmental
pattern, developmental genes were identified. See Campbell and Reece Page 415-419
N.B. Genetic analysis of Drosophila has revealed how genes control development
See the development of the fruit fly from egg cell to larva laid out in Fig. 18.19
An important point to note:
Each segment in the embryo is individually recognisable. It is possible to see if the
pattern has been altered (as in the two mutants illustrated below). But this is not
only true of the adult animal but also of the embryo: Alterations of the body plan
can easily be seen early in development as alterations of the segmental pattern in the
embryo. The pattern is disturbed so early in development that the genes mutated in
this case must act early in the process of organising the body plan
Anterior structures missing
Middle bits missing, abdominal segments.
Each of these alterations of the body plan were caused by a mutation in a single gene
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Drosophila mutant screens to identify genes involved in determining the body plan
(positional determination) was largely the work of three people and their research groups:
Ed Lewis (1950s), C. Nusslein-Volhard and Eric Wieschaus (1970s)
They were jointly awarded the Nobel Prize for Medicine or Physiology in 1995.
Their work together saturated the genome and identified more than 100 genes involved in
positional determination.
By describing the exact phenotype of a mutation and at what stage in development it
manifests itself, mutations (and the genes they represent) could be classified.
Putting all their work together they were able to group all genes involved in pattern
formation into three categories.
Maternal effect genes (axis establishment): Early acting. Genes that are
active in the mother not the embryo, so the gene product is supplied to the egg/early
embryo by the mother. These set up the major axes of the embryo (anterioposterior
(AP) and dorsoventral (DV)) early in development.
Segmentation genes (establishing segmentation pattern): These are among
the first genes in the embryo’s DNA to be turned on (expressed). They are switched
on by signals produced by the maternal effect genes. Their collective job is to divide
the embryo into segments along the AP axis.
Homeotic genes (Identity of body parts): These respond to signals produced
by segmentation genes, are expressed in a particular segmental pattern (each
segment expresses a characteristic set of homeotic genes) and determine segment
identity.
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Maternal effect genes
See Campbell and Reece Page 417 (axis establishment)
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These maternal effect genes are also called egg-polarity genes, because they
control orientation of the egg and major axes of the embryo.
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One group of genes sets up the anterior-posterior axis, while a second
group establishes the dorsal-ventral axis.
The products of maternal effect genes (proteins and mRNA) are placed in the
egg while it is maturing in the ovary. They are not uniformly distributed
through the egg cytoplasm so they act as cytoplasmic determinants (remember
lecture 9) setting up positional differences in different parts of the egg where
subsequently different daughter cells will form.
•One such gene is the bicoid gene, needed for forming the front half (head) of the
embryo. In the absence of bicoid (bicoid mutant) embryos with no head and
posterior structures (tails) at both ends are formed. See Campbell and Reece Fig
18.21
•Using DNA technology the bicoid gene was cloned or isolated. Drosophila eggs and
embryos could then be analysed to find out where bicoid mRNA was present.
–As was predicted, the bicoid mRNA was found at the extreme anterior end
of the egg cell.
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After the egg is fertilized, the mRNA is transcribed into proteins, which diffuse
from the anterior end toward the posterior, resulting in a gradient of proteins
in the early embryo.
Also, if bicoid mRNA was injected into various regions of early embryos,
anterior structures were formed at the site of injection.
So, to summarise the evidence related to bicoid function
1. If the bicoid gene is mutanted, this leads to no head and two tails
2. There is a gradient of proteins in the early embryo, high at the anterior.
3. As stated above, when bicoid mRNA was injected into various regions of
early embryos, the mRNA was translated into protein at these sites +
anterior structures were formed at the injection sites.
What does all this tell us about how bicoid functions?
Bicoid clearly is necessary to organise the body plan of the fly along the AP axis.
High bicoid = anterior structures
Low bicoid = posterior structures.
In other words, Bicoid is a morphogen, establishing body plan according to a graded
concentration (remember Morphogen concept from lecture 8).
So gradients of maternal molecules in the early embryo (such as bicoid) control
subsequent axis formation
The research on the bicoid gene is important for three reasons.
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It identified a specific protein required for some of the earliest steps in
pattern formation.
It increased our understanding of the mother’s role in development of an
embryo.
It demonstrated a key developmental principle (of a morphogen) that a
gradient of molecules can determine polarity and position in the embryo.
Gradients of other specific proteins determine the posterior end as well as the
anterior and also are responsible for establishing the dorsal-ventral axis.
N.B. Another very important aspect of bicoid
From molecular work we know the DNA sequence of the bicoid gene and the amino
acid sequence of the bicoid protein.
We know what kind of protein the bicoid gene encodes:
The bicoid gene encodes a transcription factor: A protein that can regulate the
expression of target genes (remember lecture 7).
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Segmentation genes
A cascade of gene activations sets up the segmentation pattern in Drosophila
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Since maternal effect genes like bicoid encode transcription factors, these can
regulate the activity of some of the embryo’s own genes.
Among the genes turned on by maternal effect genes are segmentation genes,
the genes that direct the actual formation of segments after the embryo’s
major axes are defined.
Sequential activation of three sets of segmentation genes provides the positional
information for increasingly finer details of the body plan. Mutations in these genes
disturb the pattern of segmentation in various ways. Mutantions in these genes were
also found in the search for genes that determine the body plan in Drosophila. The
segmentation mutations were divided into three groups depending on how early the
effect was noticed and how the defect disturbed the pattern.
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Gap genes act early and map out the basic subdivisions along the anteriorposterior axis.
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The three groups of segmentation genes are gap genes, pair-rule genes,
and segment polarity genes.
Mutations cause “gaps” in segmentation.
Pair-rule genes act a little later and define the modular pattern in terms of
pairs of segments.
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Mutations result in embryos with half the normal segment number.
Segment polarity genes ultimately define the segments and establish pattern
within each segment.
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Mutations produce embryos with the normal segment number, but with
part of each segment altered / replaced by some other part. So the
detailed pattern of each segment is disturbed.
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Homeotic genes direct the identity of body parts
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In a normal fly, structures such as antennae, legs, and wings develop on the
appropriate segments.
The anatomical identity of the segments is controlled by master regulatory
genes, the homeotic genes.
It was mutations in these genes that Edward Lewis focused on in the 1950s.
These genes specify the types of appendages and other structures that each
segment will form.
Mutations to homeotic genes produce flies with such strange traits as legs
growing from the head in place of antennae (see image of the Antennapedia
mutant below).
Structures characteristic of a particular part of the animal arise in the wrong
place.
Left: Normal Drosophila
head
Right: Head of an
Antennapedia mutant
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What are Drosophila developmental genes like at the molecular level: What kind of
proteins do they produce?
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In order to answer this question the genes had to be isolated (cloned).
We mentioned this for just one of the genes so far (Bicoid). This will
be dealt with in lecture 10.
Key concepts in lecture 9: THE GENETIC BASIS OF DEVELOPMENT
•Genetic analysis of Drosophila revealed how genes control development
•The existance of developmental regulatory genes was revealed by the study of
mutations in these genes
•Highly organised mutant screens were used to induce mutations in all the genes
that influence development (saturation mutagenesis)
•Analysis of the genes that were identified revealed that they could be grouped
according to when they acted and their actual phenotype. There are three major
groups
o 1. Maternal effect genes: First, gradients of maternal molecules in the
early embryo control axis formation
o 2. Segmentation genes: A cascade of gene activations sets up the
segmentation pattern in Drosophila
o 3. Homeotic genes direct the identity of body parts
Lecture 9: Learning outcomes: you should be able to….
A) Describe how the first developmental regulatory genes were found using
mutagenesis of fruitflies- Drosophila.
B) Describe the kinds of mutant flies that were discovered, how they were classified
into groups (maternal effect, segmentation and homeotic genes) and how these
groups showed that the body plan of the fly is established progressively. Describe
how each of the groups of genes contribute to establishing the body plan
C) Use bicoid as an example of a maternal effect gene and describe experiments that
show its important role in establishing the body plan of the fly. Link to the
concept of cytoplasmic determinants dealt with in lecture 9.
Key terms to be familiar with: Body plan, mutagenesis, maternal effect genes,
segmentation genes, homeotic genes, bicoid, cytoplasmic determinant, early embryo as
multinucleated single cell, transcription factor, anterior-posterior axis = head-tail axis,
egg polarity genes = maternal effect genes.