Developmental Biology BY1101

P. Murphy
Lecture 10
Master Regulatory genes: The genes that control development.
This lecture dealt with

Identifying developmental regulatory genes at the molecular level: What kind of

molecules do the genes that guide Drosophila development encode? How might
they carry out their functions?

Are there similar genes in other animals?

It opened the question of what the similarities and important differences are
between development in fruitflies and humans?
__________________

So what are developmental regulatory genes? We began by considering the following as

a possible classification of all genes in the genome:
(1) Structural genes
(2) Housekeeping genes
(3) Regulatory genes.
The structural genes provide the building blocks for the organism (or house as analogy).
The housekeeping genes look after the everyday running of the organism.
The regulatory genes are the genes needed to govern the construction of the organism.

These genes could be called The genetic toolkit for development

_______________________________________________
In lecture 9 you heard about the classical genetics approach and the mutational
screens that first identified the presence of developmental regulatory genes in the
fruitfly Drosophila.

They found a number of different Catagories of Drosophila developmental genes

(with sequential activity):

Maternal effect genes

Segmentation genes
1. Gap genes
2. Pair rule genes
3. Segment polarity genes

Homeotic genes

other Drosophila genes (e.g. structural genes)

Hierarchical relationships often exist between genes across groups e.g. maternal effect
genes control segmentation genes. Within segmentation genes, particular gap genes might
control particular pair-rule genes etc.

But the mutational screens that identified the genes above didnt automatically place the
genes in the hands of the researchers. Rather, they showed the existence of the genes and
their approximate location on the genetic map.
The next step was to actually isolate the genes.
The burning question at the time was, what kind of proteins are produced by genes that
have such a profound effect on the development of the body plan.
Among the first genes to be cloned (physically isolated and reproduced in the laboratory)
were 2 homeotic genes.
This was achieved using biochemical techniques and the knowledge of their approximate
location in the genome.
Note: The first Drosophila developmental genes to be isolated (cloned) were cloned
based on known position on the genetic map (their position within the genome).

When these first homeotic genes were cloned and analysed, it was seen from their DNA
sequence that they encoded transcription factors, proteins that can regulate the expression
of other genes.
Each gene contained within it, a similar DNA sequence of 180bp that encodes a 60 amino
acid domain in the protein that can bind to and regulate the control regions of other

(target) genes. This region of DNA was given the name the homeobox. The domain
within the encoded protein is called the homeodomain.

The homeodomain has a structure of three helices that bind directly to regulatory
sequences of target genes influencing their expression

Homeodomain Protein bound to DNA of

target gene

One alpha helix fits neatly into the major groove of the DNA double helix.
Other more variable parts of the overall protein (outside the homeodomain) determine
what other proteins it interacts with how it and will influence gene transcription.
___________________________________
Not all homeobox-containing genes are classified as homeotic genes: Other classes of
Drosophila developmental genes also have homeobox sequences.

The maternal effect gene bicoid encodes a homeodomain transcription factor.

The products of many segmentation genes are also transcription factors.
BUT NOT ALL
Other segmentation gene products (proteins) operate more indirectly-

Some are components of cell-signaling pathways and are involved in cellcell communication (lecture 8)- therefore instructing neighbouring cells
to change the genes they express.
This is particularly true of the later acting segmentation genes- segment polarity
genes. It makes sense that the later acting segmentation genes could act through

producing signaling molecules and receptors as well as transcription factors.

Why?
-At early stages of segmentation there are no cell membranes so no need for cell
signaling. After cellularisation, systems are needed to signal between cells to
ultimately define and refine the segmental units.
So
The genes that direct Drosophila development, when cloned, were found to encode
1. Transcription factors that regulate the expression of sets of target genes:
Homeodomain transcription factors.
Other types of transcription factors that use different protein motifs to bind
DNA and regulate transcription.
2. Signalling molecules that could be responsible for cell to cell communication.
3. Other components of signalling pathways (e.g. receptors) also used to
mediate cell communication.
Once the genes were cloned, their expression patterns in the embryo could be
investigated by in situ hybridisation. This revealed that segmentation genes are
expressed where segments will form, appropriate to their mutant phenotype.
A gap gene; the expression of Krupple

A pair rule gene: the expression of hairy

A segment polarity gene: the expression of engrailed

Homeotic genes are like other developmental genes in encoding transcription factors
that control the expression of specific target genes. In the case of homeotic
genes they must control genes responsible for the generation of anatomical
structures (either directly or indirectly).
We can use this to explain homeotic mutations.
For example, the fly segment T2 (thoracic segment 2), expresses a particular
transcription factor which identifies or labels the cells as T2. This is involved in

turning on the genes needed for the production of legs on this segment.
In a mutant, the T2 identifier gene might be expressed incorrectly in a head
segment, inappropriately labeling this segment as thoracic instead of head
and causing the eventual production of legs in the place of antennae.
___________________________________________
When the expression of the cloned homeotic genes was investigated it was found that
the genes are active in a sub set of segments where they contribute to segment
identity (i.e. they dont work alone but in combination).

Many of the homeotic genes were found to

be clustered together in the genome

Segment identity is determined by the unique subset of homeotic genes expressed in each
segment.
They encode homeobox transcription factors of a particular type and these
important genes (remember what happens if they are mutated) were given the name
Hox genes
The cluster of Hox genes also showed the phenomenon of Colinearity: Position of the
gene in the cluster reflects position along the AP axis where the gene is expressed (and
position where the gene functions). The reason for colinearity is not fully understood but
is a consequence of the mechanism of regulating where in the embryo the genes are
expressed.
_________________________

Accessing developmental genes in other organisms

Because the homeobox sequence was found in several Drosophila developmental genes,
its importance during development was very quickly realised:

Perhaps it is found in several different developmental regulatory genes because it is of

key importance to developmental mechanisms i.e. it has been perfectly conserved
through evolution in different genes. If so, then perhaps similar sequences exist in
developmental genes in other organisms i.e. the sequence has been conserved in
developmental genes in different species that arose from a common ancestor because it
plays such an important role during development.

Molecular techniques were used to search for similar genes in other organisms.
However, nobody could have imagined in advance, the extent to which these genes
have been conserved during evolution.

Homeobox genes have been highly conserved during evolution

Homeobox related sequences are found even in yeast and plants as well as all
animals, so it is a very ancient type of regulatory gene.
The homeotic genes of Drosophila that include the homeobox, fall into a
particular category of homeobox genes- the Hox genes.
Hox genes are a subset of all homeobox-containing genes particularly
involved in positional information which are also clustered in the genome.
Genes very similar to these homeotic genes (Hox genes) in Drosophila are
found in all animals from the simple jelly fish to the human, and all levels of
complexity in between.

Hox genes are found in all animals with an organized body plan
This means that the Hox genes appeared with the task of organising the body plan of an
animal (Hox genes as a subclass of homeobox genes, are not present in yeast, only in
animals with an organized body plan i.e. head end distinguished from tail end etc).
They are so valuable that they have been conserved in animals for hundreds
of millions of years.

Hox genes are a particular subfamily of homeobox containing genes that evolved together
with a complex multicellular body plan.
They have been utilised to convey positional information and organise the body plan
3 Features of Hox genes.
1. They contain a sub-class of highly conserved homeobox sequences, so they
encode transcription factors.
2. They are involved in organising the body plan of an animal.
3. They exist in clusters of similar genes in the genome.
As shown above, Drosophila Hox genes are clustered in the genome.
Vertebrate Hox genes are also clustered. See Fig. 21.18 Campbell and Reece
In mammals there are 4 clusters.
Mammalian clusters can be aligned with the fruit fly cluster based on
most similar sequence.
So:

DNA sequence is conserved

And chromosomal arrangement of the genes is conserved.
They have also conserved the order and relative position along the AP axis
of the embryo where they are expressed and function (colinearity)
The genes are in fact so closely similar that the mouse version of one gene has been
transferred to the fly and could rescue the effect of a lethal mutation in the flys own
gene.
But when Hox genes are mutated in vertebrates like the mouse, the dramatic phenotypes
seen in the fly are not produced (i.e. no mutant mice with legs on their head).
But neither do mice nor humans have a fully segmented body plan; it is more complex.
One place where we see segmentation in mammals is in the vertebral column: The
identity (shape and size) of each vertebra depends on its position along the head to tail
axis. This is where one can see more subtle transformations of identity in mouse Hox
gene mutants, like changes in the shape of vertebrae: vertebrae being produced in one
position that are appropriate to another position (see example below)
An extra thoracic vertebra
with ribs is formed when
the Hoxc8 gene in the
mouse is removed (on the
right).

So Hox genes in vertebrates also play major roles in determining the body plan
during development
Hox genes are also conserved at the functional level, they carry out similar functions
in mammals in determining the body plan as they do in the fruitfly. Not surprisingly the
body plan is more complex in mammals; more genes are needed to organize it and so the
same dramatic changes are not seen when a single gene is mutated in the mouse. If
several of these genes are mutated the individual generally does not survive.

How do they function?

They appear to operate by conferring a positional code: The Hox code.
There are 39 Hox genes in the mouse and human, arranged in 4 clusters. These are
expressed in overlapping domains along the A/P axis of the embryo,

so position along the A/P axis could be established and distinguished by the combination
of Hox genes expressed at that particular point.
This in turn determines what structures are formed there.
Such homeodomain transcription factors probably regulate development by coordinating
the transcription of batteries of developmental genes, as simplistically illustrated in
the figure below
Different combinations of homeobox genes are active in
different parts of the embryo and at different times, giving
a distinct molecular pattern to each cell type according to
its position (positional information and pattern formation).

Not only homeodomain proteins are similar, but many of the other molecules and
mechanisms that regulate development in the Drosophila embryo, have close
counterparts throughout the animal kingdom.
These fall into a number of families of conserved genes and proteins:

As for Hox genes, access to these other important regulatory genes in man was gained
through knowledge of their counterparts in Drosophila
_______________________________
There are clearly major differences between how a fly and a mouse develop.
For example, maternal cytoplasmic determinants do not appear to map out the body plan
in a mammal like the mouse.
Therefore, the fact that these regulatory genes are so similar was a major surprise.

What is particularly interesting is to understand how very similar genes guide the
development of such different organisms.
___________________________
Key concepts in lecture 10
1. The existance and many features of developmental genes were revealed by
Drosophila genetics.
2. More than 100 genes are responsible for laying down the Drosophila body
plan.
3. The first developmental genes were cloned in Drosophila and shown to
encode transcription factors- the first Hox genes
4. Drosophila genes allowed access to vertebrate developmental genes because
of the extremely high level of conservation of these genes during evolution.
5. Developmental regulatory genes do not all encode transcription factors- in
addition to transcription factors that control gene expression directly some
encode cell signalling molecules that allow cells to communicate.
6. These genes perform such important functions that they have changed little
through hundreds of millions of years of evolution (they are highly
conserved).
7. Hox genes map out the body plan in all animals from jelly fish to humans.

Lecture 10 Learning outcomes: you should be able to.

A) Describe how the discovery of developmental mutations in the fruitfly Drosophila led
to the isolation of developmental regulatory genes in Drosophila and other animals
including humans.

B) Define a master control gene or developmental regulatory gene, list what kind of
proteins they encode and how we might explain the effect of mutations in these genes on
the body plan of the animal (relate this back to the concept of positional information
(lecture 7).
C) Describe what Hox genes are, listing their 3 defining features.
D) Present the similarities and differences between Hox genes in the fly and Hox genes in
a mammal such as the mouse or human. Include a comment on the kinds of effects
caused by mutating Hox genes in the fruitfly and in the mouse.
Key terms to be familiar with: master control gene, developmental regulatory gene,
homeobox, homeodomain, Hox genes, (in common with earlier lectures: signalling
molecule, signal receptor, transcription factor), gene cluster, colinearity, gene
conservation during evolution, vertebral column/vertebrae, thoracic vertebrae, lumbar
vertebrae, change of identity in a particular position, the Hox code, combinations of
genes active