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"Your children are not your children.
They are the sons and daughters of Life's longing for itself.
They came through you but not from you
And though they are with you yet they belong not to you."
--- Kahlil Gibran
Got eyes in the back of the head
The human body is a beautiful creature.
The nudists would not like to cover their bodies with any clothes. The
strippers are proud of showing the seductive power of their naked bodies and rhythmically
movements.
Frankly, many men and women feeling proud of their nice bodies as well and
one of the most attractive feelings between lovers or partners is based on touching each
other's bodies.
However have you ever thought of why the bodies of all normal human
beings are organized in the same pattern, for instance, with the head always growing on
the shoulders, with two legs supporting the body and the eyes always on the front of the
head but not on the back.
What is the secret of controlling the development of one's body?
Beauty of the Drosophila model
To discover the secret of the body plan, scientists used insects as a
model.
The fruitfly Drosophila melanogaster became one of the favourite
models in the studies of genetics and developmental biology.
This fruitfly is a little insect about 3mm long, with a short life
cycle of just two weeks. It is cheap and easy to handle in large numbers.
In comparing with the human complexity, the fruitfly has only 4 pairs of
chromosomes (The human has 23 pairs of chromosomes); the size of its haploid genome is
about 165 million bases which contains estimated 10,000 - 17,000 genes (Human genome has
about 3,000 million bases and contains estimated 50,000 - 100,000 genes).
To observe the development process of a fruitfly, from a fertilized fly
egg passing through three larva stages and becoming an adult fly, only takes about 9 days.
(How long does it take a human being growing from an embryo to an adult?)
The Drosophila has been used as a model organism for biological
studies for almost a century and several thousands scientists are still working on it in
today's world.
As a result, scientists have created a large number of Drosophila
mutants with defects in any of several thousands genes.
When you enter the Drosophila world, it is amazing that there are
so many different morphological mutants.
To date, the fruitfly is an excellent model and the only organism that
scientists are able to begin with certain genes active in the egg and follow the
morphological development and gene activation through to its adulthood.
The beauty of the Drosophila model was first shown by its
polytene chromosome.
When the fly larva grows, its cells get bigger, each chromosome divided
hundreds of times and all the strands attached together, to form a massive polytene
chromosome.
The polytene chromosome has a unique pattern of dark and light bands
which can easily be seen under the mocroscope.
By reading the bands the whole genome can be divided into 102 numbered
bands (1-20 is the X chromosome, 21-60 is the second chromosome, 61- 100 is the third
chromosome and 101-102 is the chromosome 4).
Each of hese bands is divided into 6 letter bands (A-F). On average, a
letter band contains about 300 kb of DNA and 15-25 genes.
By reading the polytene bands, you can see any large deletion or
rearrangement of the chromosome, and individual genes can be identified on the chromosome
easily.
The thorough studies of the fruitfly have lead to two Nobel Prize
awards:
1933 Nobel Prize in Physiology or Medicine was awarded to Thomas Hunt
Morgan, for his discoveries conserning the role played by the chromosome in heredity.
1995 the prize was shared by three scientists, Edward Lewis, Christiane
Nusslein-Volhard and Eric F. Wieschaus, for their discoveries concerning the genetic
control of early embryonic development.
On the excellent basis of their work, the fruitfly has provided us the
most complete picture of all species of how the genes controlling the development
of the body.
Scientists are then be able to show the magic power of genetic
engineering: by manipulating certain master controlgenes they have produced a numbers of
"Modern Frenkeleins" and truly some of those flies have extra eyes on the
back of its head.
Modern Frenkeleins created by genetic engineering
Legs grew on the head
The story goes back to 1987, Dr. Walter Gehring and his group in
University of Basel, Switzedland.
They had put a gene called Antennapedia
into the head of the fruitfly and forced it to be active.
The Antennapedia gene was normally
expressed in the throacic and abdominal parts of the fly body, and it controls that part
of the body plan, where legs were made on each segment.
Once the Antennapedia gene was
expressed in the wrong place - in the head but not in the thorax -, then the normal body
plan was changed.
The consequences were the formation of middle legs in the place of the
antennae and the second thoracic segment in the place of the dorsal head capsule.
So the wrongly placed legs extended out from the head, making that fly
really looked like a "Modern Frenkelein".
The Antennapedia gene belongs to a
group of genes called Homeotic genes, they specify the development of individual segments
of the fly.
In other words, this group of genes are commanders of controlling the body
plan.
They interact with each other nicely but everyone has its own working
space and different reponsibilities.
The Antennapedia gene was
responsible for the specification of the midbody. When the scientist put the Antennapedia gene in a wrong place, it sent out commands
telling the head to be developed as the midbody and that including the formation of legs
instead of antennae.
In this case, the scientists have proved that the body plan was truly
determined by the genes.
"Eyeless", a master control gene?
However the investigations continues, in 1993, Gehring's group cloned
the eyeless (ey, pax6) gene of fruitfly and discovered
that eyeless gene is conserved in mice and human.
The mouse version is called Small eye (Sey, pax6)
and the human version is called Aniridia (AN2, pax6).
It was a surprise, because the structures of eyes have been very
diversified in the animal kingdom, from simple light-sensitive photoreceptors to complex
camera-like eyes.
The insects' compound eyes are so different to the single-lens eyes of
vertebrates.
There was a hypothesis that the eyes of different species had distinct
evolutionary origins, and the common photoreceptors in their structures may have evolved
independently 40 - 60 times during the evolution.
However an alternative view suggesting a common ancestor of the various
eyes.
Scientists wondered if this eyeless
gene could be conserved through more than 500 millions years evolution process from insect
to human, that means it might be very important to the development of the eyes - possibly
it belongs to "master control genes".
The highly conserved genes usually are the favourites for our geneticsts, because of the million years natural evolution they have selected them for the perfection or survival of the organisms.
As a matter of fact, the eyeless mutation of Drosophila was first described in 1915 by M. A. Hoge in Am. Naturalist, based on its characterictic phenotype: the partial or complete absence of the compund eyes. However the detailed analysis was not possible at that time.
After almost 80 years, scientists finally reached the stage of understanding more about the genetic reason of this mutation and cloned the eyeless gene.
Today's cloning technology made it possible to manipulate the gene and to explore it functions and power. Gehring and his group designed an interesting experiment to test the power of the eyeless gene.
The life cycle of the fruitfly
The body of a adult fly consists of head, 3 thoracic segments, 8 or 9
abdominal segments and tail.
The development of a adult fly from a fertilized egg passing through
several stages:
The Drosophila egg is long and narrow with a size of 0.4 mm x 0.16
mm.
The embryonic development starts immediately after the fertilization. The
egg divides rapidly to form 2, 4, 8 . . . cells.
After the 16-cell stage, cells begin to specialize in the embryo.
It takes about one day after fertilization for the embryo to develop and
hatch into a worm-like larva with 14 segments appears (Each segment in the larva results
in a specific segment in the adult fly).
The larva eats and grows continuously for 5 days, moulting through three
larval instar phases.
Then passes into the pupal stage, in the immobile pupa, most of the larval
tissues die and are reabsorbed; the remaining larval tissues are specilized groups of
cells so called "imaginal discs" (these imaginal discs develop into the organs
of the adult fly).
After some days of pupation and growth, metamorphosis takes place.
The main part of the head, including the frons, the antenna, the eyes and
the maxillary palps are formed from the eye-antennal disc.
The major part of the thorax, including the wing, notum, scutellum and
pleura are developed from the wing disc.
The legs are formed from the legs discs and the haltere is formed from the
haltere disc. Finally a new fly emerges.
Drosophila biologists have been working on these imaginal discs
for many years. They can isolate and remove individual imaginal discs from the larvae of
the fruitfly and induce them to develop in culture - To follow the formation of the organs
: wing discs form wings, leg discs form legs and so on.
These imaginal discs usually work very well and loyally to follow their
specilized destiny.
However occasionally the development of an imaginal disc could go wrong -
the wing disc cultures turned out not forming the semi- transparent wings but showing the
brilliant red pigmented facets. This phenomenum called transdetermination.
To express eyeless in wrongly place
Is it possible that the transdetermination of the organ development was
caused by the accidently switch-on of certain master control genes?
With the eyeless gene in their hands,
Gehring and his group tried to make it express in different imagical discs rather than the
original eye disc - If the eyeless gene is the master control gene for eye formation, it
should be able to induce eye structures in other parts of the body.
They utilized a strategy called "targeted gene expression
system", to restrict the location of areas of the exotic eyeless
gene expression (See Figure: "Genetic Engineering Produced
Supernumerary Eyes Fruitfly").
How did they do it?
First of all, they established two different types of genetic engineered fruitfly stocks.
The flies in the first stock have a GAL 4 sequence inserted into their
chromosome. (This GAL 4 originally came from yeast studies, it can activate any gene after
introduction into Drosophila, if that gene possesses a upstream GAL 4 binding site).
These GAL 4 lines start to express GAL 4 protein during embryonic stages.
A GAL 4 line E132 was chosen from the first stock to demonstrate the
expression of GAL 4 protein in discrete regions of the wing disc, antennal discs, haltere
disc and all 3 pairs of leg discs.
The flies in the second stock have a extra eyeless gene under the control of a upstream activating sequence (UAS, which consists of 5 GAL-4 binding sites). The extra eyeless gene in these UAS-ey lines of the second stock on it own was not active; its expression can only be switched on when the GAL 4 protein meets the URS site and binds to it.
They took the GAL 4 lines crossing with the URS-ey lines, transheterozygous flies were generated, and the expression of the extra eyeless gene was targeted into the distinct regions of the imaginal discs where the GAL 4 was expressed. In the wild type control fly the eyeless gene was only expressed in the eye portion of the eye-antennal disc.
Eyes grew all over the bodies
As the consequence of this ectopic targeted expression of the eyeless gene, supernumerary eyes were grown all over the flies'
bodies.
Those extra eyes were in the wings, all six legs, the halteres and even on
the ends of the antennae that looked like little crab eyes. Some flies had as many as 14
eyes!
Were these supernumerary eyes possessed of normal eye structures and can function as normal eyes?
The compound eyes of the fruitfly consist of about 800 optical units
called facets or ommatidia.
Each ommatidium contains 8 photoreceptor cells which associated with
rhabomeres, one through six of the photoreceptor cells are placed radially around cells 7
and 8. Each ommatidium is surrounded by two primary pigment cells, and these are
surrounded by six second pigment cells.
Thus each ommatidium contains a total of 22 cells, making the total number
of cells in a fly eye over 16,000 cells.
This type of eyes exist in Arthropods such as crustaceans, spiders and insects, since each ommatidium of a compound eye is directed at a slightly different part of the visual field, only a crude image of the visual world can be transduced to the central nervous system.
The fine structure of the ectopic eyes induced by the eyeless gene was analyzed by scanning electron microscopy.
The large, well developed ectopic eyes were mostly seen on the wings and
antennae, the array of facets and bristles were largely normal, however in some cases
fusion of facets and irregular spacing of bristles were observed.
The eyes induced on the legs were on average smaller, but nevertheless
appeared with the normal structure.
Microscope analysis of sections of ectopic eyes showed it has the full
complement of the structures and cell types of a compound eye, including cornea,
pseudocone, cone cells, primary, secondary and tertiary pigment cells, and photoreceptors
with rhabdomeres.
The normal trapezoidal array of rhabdomeres was clearly visible. Clusters
of photoreceptor cells were detected at the ectopic sites, and the sequence of neuronal
differentiation was retained in the ectopic eye cells.
The photoreceptors in the ectopic eyes were electronically active upon
illumination.
These evidences showed that the eyeless
gene induced the formation of complete and morphologically normal ectopic eyes.
However it was unknown whether the axons of the photoreceptors connected
to the correct domains of the brain.
Mouse Small eye gene doing the same job in the fly
Another question asked by the scientists was the relationship between the Drosophila eyeless gene and its counterpart genes in mouse and human.
The single-lens eyes of vertebrates are functioning like cameras and
poss exceptional ability.
If the mouse Small eye (pax6) gene is
put into the fly system, what will happen?
In the mouse, Small eye is expressed
first in the optic cup, then in the lens and finally in the cornea.
It might be the master control gene in the mouse eye development process?
They put the mouse Small eye gene into Drosophila, did the same targeted expression experiment.
The result was that the mouse Small eye
gene can also induce the formation of ectopic eyes in fruitfly.
Fortunately, the ectopic eyes induced by the mouse gene still contained
the structures of Drosophila type eye, but not the mouse eye.
This study (Published in Science, 267:1788 - 1792, 24 march 1995)
indicates that the genetic control mechanisms of body development are much more universal
than anticipated.
Is it possible that the insects and mammals are using the same master
control gene for eye morphogenesis?
Squid pax6 gene induced ectopic eyes in the fly
Recently another extending result was published (Proc. Natl. Acad. Sci.
USA (94: 2421- 2426, March 1997).
Tomarev et al in National Eye Insitute of NIH, Bethesda, in cooperation
with Gehring's group to introduce another systematic type of eyes into the scene.
They have cloned the pax6 gene of Squid and shown that it is expressed in the embryonic eye, olfactory organ, brain and arms of the Squid Loligo opalescens.
Cephalopod molluscs (squid, octopus, cuttlefish) have a well developed nervous system and are quite intelligent, their eyes are remarkably similar to the vertebrates' in the appearance and organization but are formed by different mechanisms during development.
Because there are no such excellent research facilities for the functional studies of the Squid pax6 gene in Squid, they first tested it in the established Drosophila targeted expression system which we have mentioned above.
Interestingly enough, the Squid pax6 gene also can induce ectopic Drosophila eyes on the wings, antennae and legs of the fruitfly.
Those repeated demonstrations of the evolutionary conserved and critical role of pax6 gene in eye development from insects to molluses and mammals is therefore being taken seriously.
Genetic regulatory hierarchy of the eye development
How could a single gene like the eyeless ( pax6) gene have such dramatic power to determine the formation of a whole organ?
Actually it is not working alone, there are at least 2,500 genes
involved in the development of the eye in Drosophila.
Eyeless luckily is at the top of the
regulatory hierarchy of the eye development.
Once the eyeless gene is switched
on, it triggers a set of subordinate regulatory genes to start work, then the signal
transduction pathways, cell-cell interactions are subsequently entering the cascade.
Finally the structural genes like rhodopsin, crystallin and transducin
must be expressed to build up the eye structure.
Scientists have studied more than hundred of genes involved in Drosophila
eye morphogenesis. For instance, eyes absent, sine oculis, eyes
gone and eyelish, these genes showed
similar phenotypes.
However they could not affect the expression pattern of eyeless, indicating that these genes are downstream or act
parallel with eyeless.
Shen et al (Development 124: 45-52, 1997) in Baylor College of Medicine, Houston, studied another gene called dachshund (dac) showed that targeted expression of dachshund directed ectopic retinal development in the head, thorax and legs of the flies.
Dachshund and eyeless induce the expression of each other, and dachshund is required for ectopic retinal development driven by
eyeless.
However dachshund may still functions
downstream of eyeless, the evidences were:
(a) Expression of eyeless in the wing,
leg and antennal discs can induce ectopic dachshund
expression in all discs.
(b) When dacshund was mutated, eyeless was still able to express but could not induce the
formation of ectopic eye.
These results suggest that the control of eye development requires the complex interactions of multiple genes, even at the top of the regulatory cascade.
Another new challenge gene might be six. This gene was discovered in the mouse, its sequence is similar to Drosophila sine oculis, it expresses in the developing eye regions and is one of the most anterior homeobox genes reported to date.
Six3 expresses normally in Small eye (pax6) mouse mutants.
That means six3 may function upstream
of pax6.
However there has been no demonstration of misexpression of pax6 in any vertebrates that can induce ectopic eyes. Perhaps
the regulatory mechanism of eye development in vertebrates is much more complex than it is
in fruitfly.
At the present time, we human beings don't need to worry about the possibility of having supernumerary eyes on the back of our heads.
Nevertheless, the Drosophila "Modern Frenkeleins"
experiment is a stunning demonstration of using the powerful genetic engineering
technology to modify the body plan of organism.
Genes control the body plan
- Nobel prize laureates 1995
1995 Nobel prize in Phsiology or Medicine awarded to three Drosophila
scientists, Edward B. Lewis, Christiane Nusslein-Volhard and Eric F.
Wieschaus, for their excellent achievements in the genetic control of
early embryonic development.
How do the genes control the body plan of animals and ourselves?
These scientists have used Drosophila melanogaster as a model
system, systematically disclosed the secrets of how could the genes determine the body
plan of Drosophila from its embryo to an adult fly.
The principles found in the fruitfly apply also to higher organisms including human beings.
Key genes discovered from ~40,000 mutants studies
Nusslein-Volhard and Wieschaus were two young scientists at the
European Molecular Biology Laboratory (EMBL) in Heidelberg at the end of the seventies of
20th century.
They both were interested to find out how the fertilized Drosophila
egg developed into a segmented embryo.
They made a brave strategy to start this exploration.
They treated flies with chemicals to mutate about half of its genes
randomly, so called "saturation mutagenesis".
They thought that since genes control embryonic development, it should be possible to see the genetic damages resulting defects in the offsprings and thus identify genes that specify segmentation - the genetic determination of the body plan.
For more than a year the two scientists sat opposite each other using a
microscope which they could simultaneously examine the same embryo.
They examined about 40,000 mutations of embryos resulting from genetic
crosses of mutant strains.
Finally they identified and classified 15 genes of key importanmce in determining the body plan and the formation of body segments of the fruitfly (Today the gene numbers have increased to 25 or more).
These genes are divided into 3 functional groups :
The gap genes lay the rough body plan along the head-to-tail
axis.
The pair rule genes govern the formation of every second
body segment.
The segment polarity genes refine the head-to-tail polarity
of individual segments.
Four wings fly leading to "co-linearity principle"
Edward Lewis at the California Institute of Technology in Los Angeles has been studying the genetics basis for homeotic transformation in Drosophila since the forties of 20th century.
Already geneticists had noticed some odd malformations in Drosophila.
In one type of mutation the body plan was disturbed to grow an extra pair of wings in the
place of the halteres.
The Greek word "homeosis" was used to describe such type of
malformations - it means that something has been changed into the likeness of something
else - and the mutations were referred to as "homeotic" mutations.
The famous little monster fly with four wings interested Lewis.
He found that the extra wings came from the duplication of a body segment
- the second thoracic segment - that was caused by the mutation of a memebr gene in the
Bithorax complex gene family (We will look into this gene later).
Lewis has worked on homeotic genes and segment specialization for
decades and was far adead of his time.
In 1978 he published his famous theory of "the co-linearity
principle".
There are two homeotic genes complexes (Antennapedia complex
consists of 5 or 6 genes; Bithorax complex consists of 3 genes) in Drosophila
to control the development of a larval segment into a specific body segment.
He discovered a co- linearity in time and space between the order of the
homeotic genes and their effect regions in the body segment.
These genes all contain a conserved domain, so named "homeobox".
Lewis's pioneering work induced the next wave of discoveries :
In vertebrates including humans there are similar gene families called hox
family. Human genome have four hox gene complexes, they occcur in the same order
and exert their functions along the body axis in agreement with the co-linearity
principle.
Five Gene groups control the body plan
Now let us examine the exact developing process of genes controlled body development of Drosophila which gives us the most complete picture in this field and no other model system has been able to catch up yet.
The genes determine the body plan in a fruitfly can be divided into 5 major groups according to their order of entering the stage and their individual tasks:
(A) Maternal co-ordinate genes:
bicoid, nanos, torso etc.
(B) Gap genes:
buttonhead, cap'n'collar, candal, empty
spiracles, giant, hunchback, huckebein, Kruppel, knirps, odd-paired, orthodenticle, sloppy
paired, tailless etc.
(C) Pair rule genes:
Even-skipped, fushi tarazu, hairy, odd-paired,
odd skipped, paired, runt, sloppy paired, Tenascin major etc.
(D) Segment polarity genes:
Hedgehog, wingless, engrailed, invected, cubitus
interruptus, decapentaplegic, deformed, patched, ovo, lady bird early and lady bird late etc.
Genes dowstream of hedgehog: costal-2, cubitus interruptus, decapeentaplegic, engrailed, fused, naked, patched, sloppy paired, smoothened, wingless.
Genes downstream of wingless: armadillo, decapentaplegic, dishevelled, engrailed, frizzleed2, gooseberry, gooseberry proximal, porcupine, shaggy/zeste white 3.
Genes downstream of dpp: aristaless, decapentaplegic, mother against dpp, optomoter blind, punt, saxophone, schnurri, spalt, thick veins, wingless.
(E) Homeotic genes:
Antennapedia complex genes:
Labial, proboscipedia, zerknullt, bicoid,
deformed, sex combs reduced, fushi tarazu, antennapeedia. (bicoid and fushi tarazu are
located in the same cluster, but they are classified as Maternal co-ordinate gene and Pair
rule gene respectively).
Bithorax compleex genes:
Ultrabithorax, abdominal-A, abdominal-B.
Mateernal coordinate genes (Bicoid, nanos, torso etc)
These genes are involved in patterning the egg while it lies within the
ovary.
They determine the anterior/posterior (head-tail) and dorsal/ventral
(back-front) axes of the embryo.
At the very beginning stage, the body plan starts off with genes that are expressed by the maternal genome, the nurse cells in the ovary synthesize and deposit their RNAs into the deeveloping egg.
The bicoid mRNA is deposited at the
anterior tip of the egg, then the mRNA starting traslation from there to form an gradient
of the bicoid protein.
Bicoid is a transcription factor and
plays an essential role in establishing the anterior/posterior axis.
Its gradieent acting to position the expression of gap genes and pair rule
genes along the anterior/posterior axis.
Bicoid protein binds to the 5' end of the gap gene hunchback and activing it.
Hunchback becomes expressed in the
anterior part of the embryo.
But within a gradient of bicoid
protein, when the hunchback expression reached the
area of threshold low level of bicoid, it had to stop
expression.
Hence the expression of hunchback is located to the anterior half of the embryo, and it is stopped in a discrete line.
When the bicoid gene was mutated, resulting in the embryo having no anterior thorax.
Another maternal coordinate gene is nanos,
its mRNA is deposited to the posterior end of the egg and nanos
protein formed a posterior/anterior gradient..
Nanos protein is a translation
inhibitor, it prevents the maternal hunchback mRNA to
translate into protein.
Under such double regulations, the hunchback
has no chance to express in the posterior half of the embryo.
Then it permits the next step for gap genes to specify the abdominal
region.
Nanos mutants have no abdomen, the maternal hunchback was lost control and distributes throughout the whole embryo.
Torso gene is responsible for
patterning the acron and telson at the extreme tips of the larva.
Torso mutants showed no head and tail
ends of the embryo.
Gap genes (hunchback, giant, Kruppel and knirps etc)
These genes are the egg's own genes, so called zygotically active
genes.
They are busy in the very early embryo, the syncytial blastoderm, to
specify a rough body plan, the anterior, middle and posterior parasegments.
When the gap gene was missing, it caused a large gap in the embryo.
The gap genes hunchback, giant, Kruppel and knirps acted in concert to set their individual function domains.
We already knew how hunchback expression
was regulated by bicoid and nanos,
and localized it to the anterior half of the embryo.
Hunchback plays a central role in
determining the placement of other gap genes, as it can operate both as a transcription
activator or repressor.
Hunchback itself specifies the thorax parasegment. Its mutants are missing thorax part.
Kruppel is another gap gene makes a
zinc finger protein to specify the middle part of the body plan.
Its expression forms a single broad band, like a belt, around the centre
of the embryo. It is regulated by several genes - Bicoid
activates it, knirps and giant
repress it, hunchback
actives it at low concentration but represses it at high concentration.
At the 5' end region of the Kruppel
gene, there is a 16 base pair region where both bicoid
and knirps proteins could bind to it.
The competition binding between regulatory factors is one of the gene
regulatory mechanisms.
When Kruppel gene was mutated, the embryo lacks a large part of the body, T1 - A5 segments were missing.
Knirps is responsible for the
posteriod part of the body.
Two broad stripes of knirps expression
lie adjacent to the edges of the Kruppel band.
Knirps expression is repressed by
anther gap gene tailless, but directly enhanced by Kruppel.
Thus Kruppel acticity is present throughout the domain of knirps and forms a long-range protein gradient, which in combination with knirps activity is required for abdominal segmentation of the embryo.
Knirps mutants lack posterior segments.
Giant codes for a repressor
protein.
Early in development it is expressed in two broad stripes about the two knirps domains and closer to the terminal ends.
The activation of giant involves bicoid and another gap gene caudal,
however hunchback acts as a concentration-dependent
repressor of giant.
The expression of giant in the head becomes more complex over time - developing 4 stripes in the head segment - suggesting it is involved in the process of head morphogenesis.
Giant mutants produced gaps in both anterior and posterior structures, the labial and labral head structures and abdominal segment A5 - A7.
The interactive performance of the segmentation concert by these gap genes then promote the next program, the pair rule genes.
Pair rule genes (Even-skipped, fushi tarazu etc)
The pair rule genes subdivide the embryo into 14 parasagments. (The embryo initially divided into the parasagments (numbered from 1 to 14), which are later replaced by segments (named Head, T1 - T3, A1 - A8) that are formed from the posterior part of one parasegment and the anterior part of the next one).
Even-skipped and fushi tarazu are pair rule genes like sisters.
They both are expressed in vertical stripes in the early developing
embryo.
Seven stripes of fushi tarazu are
interspersed with seven stripes of even-stripped,
forming a total of 14 evenly spaced alternating bands.
Even-stripped is expressed in even
numbered parasegments and fushi tarazu is expressed in
odd numbered parasegments.
Even-stripped is a repressor and fishi tarazu is a transcription activator.
Their complementary functions are crucial for the activation of the next
program, the segment polarity genes.
Even-stripped looks like being the old-sister, it is termed a primary pair rule gene, since its activation depends on maternal and gap genes bicoid and hunchback.
Each strip of even-stripped is
independently activated by separate specific enhancers (Enhancer is a special sequence
located nearby the structural gene and be able to enhance the expression of the gene).
For example, there is a strip 2 enhancer located 1.0 - 1.5 kb upstream
from the even-stripped gene that assure the even-stripped expressed in the strip 2 region.
It was done with help from giant
repressor which forms the anterior border of the stripe 2; and with repression from Kruppel to determine the posterior border of the same stripe.
Fushi tarazu, however, has induced some puzzles. Despite a decade of work has not yet identified stripe specific enhancers for her.
Loss of a pair rule gene, for instance, even-stripped mutants allowed only odd numbered segments to develop.
Segment polarity genes (Engrailed, wingless, hedgehog, decapentaplegic etc)
The next show is brought in by the segment polarity genes.
They make the true segments out of the parasegments, each segment is
subdivided into anterior and posterior compartments, resulting in cell identity.
Engrailed, this segment polarity gene is regulated by concerted actions from six pair rule genes to ensure it is expressed in each anterior compartments of the 14 parasegments.
Wingless is expressed in each posterior compartments of 14 parasegments.
They were right beside each other showing a very fine spatial expression pattern.
At this stage, cellularization of the blastoderm has happened, so cells start to communicate with each other. Wingless and hedgehog proetins are diffusable factors.
Engrailed activates hedgehog expression in the anterior compartment of each
parasegment, the anterior cells secret hedgehog protein
which binds to wingless expressing posterior cells,
stimulating them to make more wingless protein.
In this way, the boundaries between the and wingless
expressing cells are defined.
And finally the boundaries disappear - the true segments bounderies begin to arise.
Wingless plays a primary role in specifying the wing primordium, the ectopic expression of wingless can induce supernurnary wings in the portion of the disc normally fated to give rise to body wall.
Hedgehog defines the polarity of the parasegments fated to develop into head, thorax and abdomen.
It takes several genes acting together to do the segmental specialization of the wing: The embryonic posterior compartment of parasegment 2 combines with the anterior compartment of parasegment 3 will arise the adult wing.
Hedgehog is induced by engrailed and secreted from posterior cells, it diffuses a short distance acts on adjacent anterior compartment to overcome repression by patched gene and resulting in the activation of decapentaplegic.
Decapentaplegic then defines the compartment border between the arterior and posterior halves of the wing.
Once the segments are defined, another powerful genes group comes into action, the famous homeotic genes - the magicians.
Homeotic genes (Antennapedia complex: Labial, proboscipedia, zerknullt, deformed, sex combs reduced, antennapedia. Bithorax complex: Ultrabithorax, abdominal-A, abdominal-B)
The homeotic genes are transcription factors, they control
specialization of the segments, in other words they determine which body structure should
be arise from which segment.
(Now we are familiar with the roles of the transcription factors, they
have the power to control other genes, to induce their activation or to inhibit them, and
sometimes they trigger a regulatory cascade which could involve thousands of genes to do a
real big job in our bodies).
At this stage the larvae of the fruitfly has 14 segments.
The first three segments will become the head, the next three T1 - T3
segments will become the thorax and the rest A1 - A8 segments will become the abdomen.
Do remember there are specialized cell groups "imaginal discs"
which deferentiate to form the organs of the adult body.
That's the job of these homeotic genes, they direct the differentiations of the segments and are responsible to make sure that the imaginal discs develop properly.
The study of the homeotic genes is one of the exciting part in development biology. These homeotic genes are excellently arranged in a linkage array and go in to action one by one in a strict order, and the studies of homeotic genes by mutating them always give you the exciting visible results in the features of the bodies.
To date, there are 9 homeotic genes defined in Drosophila, they
are arranged into two gene clusters:
The Antennapedia complex consists of 6 genes, it is located on
chromosome 3 - 47.5 and the Bithorax complex consists of three genes, it is located
on chromosome 3 - 58.8.
We would start to examine some interesting characters of them.
Black Magic
Facing an alien head?
Do you believe it or not, some people said, if you come face to face with a highly amplified fruitfly head it can be easily confused with an extraterrestrial alien head.
The huge compound eyes protrude on each side of the head, between them
a pair of swollen antenna, the aristae and a sensory pouch sacculus occupied the
"forehead".
Lower down the "face", one find the "nose" - clypens,
but it neither breath nor smells.
From the bottom of the clypeus hanging down the labrum, a short tube used
for sucking food.
On each side of the labrum are two small bulbs, the maxillary palps.
Below the small bulbs lies the large furrowed bulbs - labial palps, they
lie at the end of the proboscus, the fly's main feeding organ.
These furrowed bulbs gather liquid, moving it upward through a series of
collecting channels until it reaches the hollow, straw-like labrum.
From there the food continues upward into the pharynx by muscular action.
Magic players
Labial is the first homeotic gene goes into action, as it is the most proximal one in the Antennapedia complex and expressed in the anterior of the fly.
Labial has two roles in Drosophila morphogenesis, one in the developing head, and another in the midgut.
The second member of the Antennapedia complex named proboscipedia, it is required for the formation of labial and maxillary palps.
When proboscipedia is mutated, labial palps are transformed to prothoracic legs, and maxillary palps are small and malformed.
Beside serving a homeotic "selector" function in the head, Proboscipedia also regulates other homeotic genes.
An interesting example is in the case of the ectopic leg grown in the head instead of antenna, it was induced by another homeotic gene Antennapedia.
However if we put a heat shock promoter directed proboscipedis gene in and force it to express at high dosage
until a threshold level. The Antennapedia expression
was inhibited by proboscipedia, and the ectopic leg was
switched to ectopic maxillary palp!
This proboscipedia has won the
competition with other homeotic genes to alter the fate determination.
Zerknullt is an unique homeobox
gene, it is required for the differentiation of the dorsal- ventral pattern of
Drosophila, and does not appear to be involved in the process of segmentation.
In the absence of zerknullt, germ band
extension is abnormal, it is twisted and thrown into folds.
Deformed is responsible for the
normal development of the maxillary segment, those tiby bulbs in front of the labium.
Deformed mutation also associated in
three lethal effects during metamorphosis.
Ectopic expression of deformed results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear.
Sex combs reduced is required for
labial and first thoracic segment development.
Its mutants show a tendency to transfrom labial into maxillary structures.
This gene is sandwiched between complex regulatory regions comprising a
span of over 70 kb of DNA extending from Antennapedia at
one end, sandwiching in a pair rule gene fushi tarazu on
the way and continueing to a point beyond the 3' terminus of deformed
gene.
Sex combs reduced seems at least
involveed in three areas of Drosophila development: labial differentiation, CNS
development and mesodermal development, each with independent gene regulation. The
research is still under way.
Now it's the time for the last member of this complex, Antennapedia itself to enter action.
The famous "antenna-foot" black magic
It is the one most distal from the centromere and the one expressed in
the most posterior locale.
Antennapedia is the initiator of the
development of an adult leg.
Its highest expression level is found in the second thoracic segment T2.
As early as 1947, a type of transformation mutant had been found, the mutant fly having no antenna but two sets of second legs. The name Antennapedia means "antenna-foot" was used to describe it.
The spontaneous mutant that give rise to antenna to leg consists of an
partial duplication of the Antennapedia gene as well
as a insertion of >40 kb new DNA in its genomic region.
This mutation gives this gene three promoters, and at least two of the
promoters are ectopically active in the eye-antenna imaginal discs, leading to the
homeotic trtansformation of the fly head.
Later on , the genetic engineering to express Antennapedia
gene in the head has induced the same phenotype:
The fly grew ectopic legs in place of antenna.
How could it control the transformation of a whole organ?
There might be thousands of genes downstream of its command.
One of the pathways has been studies:
Ectopic expression of Antennapedia in
the head is likely to repress a gene called spalt
which involved in promoting head and tail patterns.
Spalt normally represses another gene teashirt which is known to promote trunk (thoracic)
development.
Once spalt has been inhibited in the
head by Antennapedia, teashirt get freedom to active
in the head with the trunk identity including the formation of second legs.
Scientist then discovered a reverse transformation phenotype, null
mutants of Antennapedia let the second leg
transforming into an antenna.
It is possible that during the absence of Antennapedia,
the inhibition of other head fate determine genes was released and they occupy
the empty space of the second thoracic segment.
Playing with homeotic genes is like playing magic. In principle, depending on genotype any segment can change its identity to resemble other more anterior or more posterior segments. "Man masters nature not by force but by understanding, This is why science has succeeded where magic failed . . ." - Jacb Bronowski
Recently it has been shown that ectopic expression of the three
homeotic genes in Bithorax complex Ultrabithorax, abdominal-A and
abdominal-B, are also able to transform antenna into leg and wing into haltere.
Fruitfly challenges Butterfly
Ultrabithorax is the first gene in
the Bithorax complex, it regulates the decisions for the numbers of wings and legs.
Drosophila have only one pair of wings, butterflies have two pairs
of wings. However Ultrabithorax could make the humble
fruitfly challenges the brilliant butterfly.
There are three thoracic segments in Drosophila larva, the frist thoracic segment T1 grow the first set of legs, the second segment T2 grow the wings and the second set of legs and the third segment T3 grow the halteres and the third set of legs
Mutations of Ultrabithorax result
in transformation of the T3 segment (the halteres and third legs) into the T2 segment (the
wings and second legs).
The doublication of T2 segment producing four wings for our Drosophila
princess.
Then Drosophila said proudly to Butterfly:
"Look! I have four wings as well."
Butterfly was not convinced by her words, he said:
"Wait a moment, I have to check with my
Ultrabithorax gene to see it it can make more wings for me." And he ran to ask the
Ultrabithorax gene
However Ultrabithorax could not make Butterfly to have
more wings.
Ultrabithorax is expressed in posterior thgoracic segments in all insects including butterflies and other modern four wings insects - however none of them could get extra- wings from Ultrabithorax.
That was not the fault of a single gene, there are multiple genes and multiple changes concerted in Drosophila with Ultrabithorax to profuce a second pair of wings. In other insects, this network has not been found yet.
Butterfly then moaned: "Oh! You little fruitfly! You are the favourite of geneticists. They have done so much work on your genes and your body alterations. But we will catch up . . ."
We have no time to listen to their augment. It's better to finish our examination of the last two hometic genes in Drosophila.
Abdominal-A involved in the pattern
of cuticle generated in the ectoderm and the muscle generated in mesoderm.
With ectopic mesodermal abdominal-A expression,
the patterns of the mature thoracic longitudinal muscles changing to a more abdominal like
identity.
Abdominal-B is the last gene in
linkage order and the most posterior acting gene of the Bithorax complex.
It is critically involved in the tail development. Abdominal-B has some unique characters, making two forms of
proteins, one functions as regulator in segment A9, another functions as morphogenic
protein in segment A5 - A8.
Thus all the homeotic selector genes went into actions according the co-linearity in time and space between the order of theior positions on the chromosome and their effect regions in the segments.
Impressively enough, the type of genes that control the body plan for
the development of fruitfly also controls the early embryogenesis of a lot of higher
organisms including man.
From fly to man
What have we learned from the knowledge of genes controling the
development of Drosophila?
There are more than 500 million years evolution period between the insect
and man.
Strikingly, many developmental pathways found in Drosophila have
been conserved across a wide variety of species until ourselves,
More homeobox genes and clusters in humans
The homeobox genes we have examined above are conserved and further
developed in our own bodies.
In Drisophila there are 9 homeobox genes arranged in two complexes
or clusters.
In mouse and human there are 38 homeobox genes in 4 clusters.
In both Drosophila and humans, the homeobox clusters control the identity of cells along the anterior/posterior axis, including cells of ectodermal, mesodermal, endodermal and neutral fates.
Each homeobox gene in Drosophila has found their multiple
counterpart genes in mammals.
The labial gene homolog is found at
one end of the homeobox clusters and the AbdominalB
gene holog is found at the other end of the clusters for each and every animal which has
been looked at.
And the structurees of these genes are so highly conserved that it is possible to functionally substitute a human homeobox gene for its Drosophila counterpart gene.
The homeobox gene clusters in mouse and human are called Hox-a through Hox-d.
The genes in each cluster are numbered 1 to 13.
For instance, when the mammalian counterpart genes (HOX-B2, HOX-B3 and HOX-B4) of proboscipedia and deformed were
lost, the result is incorrect development of the branchial arches, neck or head
structures.
Drosophila lessons have been helping us to understans more about the genetic control of human body.
Important functions of pax family
Finally let us go back to look at the evolutional situation of eyeless (pax6) homolog genes family from fly to man.
To examine the protein structures of the pax6 genes from all species, finding two common domains - the functional "paired box" domain and the "homeobox" domain (Beside the homeotic genes clusters mentioned above, there are many other genes that contain a homeobox sequence.)
The eyeless gene spans
approximately 16 kb on the chromosome 4 of Drosophila.
It encodes two splicing variants of mRNA differ in their first exons,
resulting in two eyeless proteins of 838 amino acids and 857
amino acids in length respectively.
Both proteins contain the functional N-terminal "paired box"
domain (124 amino acids) and a central "homeobox" domain (60 amino acids).
These domains share extensive sequence identity (90 - 94%) with mouse and
human pax6 genes.
The pax family including the proteins contain the "paired box" domains, some do have a homeobox too, some do not have.
So far 9 members of the pax family have been identified in mouse and human:
Pax1 - located on human chromosome 20, which may play a role in the developing vertebral column.
Pax2 - located on human chromosome 10, its mutations are associated with optic nerve coloboma eith renal disease.
Pax3 - its position is on human
chromosome 2, the mutations are associated with Waardenburg Sndromes (WS) and cancer.
WS is a genetic disorder which probably influences 2-3 out of every 100
childrens in the schools for the deaf.
The child with mutations in his (or her) pax3
gene usually has wide bridge of the nose, two different colored eyes such as one is brown
and the other bright blue, or patches of brown and blue are mixed in the same eye, white
forelock, white eyelashes and premature greying of the hair, and some degree of cochlear
deafness.
Pax4 is on human chromosome 7 and showed the most diversed paired box of the family.
Pax5 has another name BSAP (B-cell lineage Specific Activator Protein), it controls expression of the CD19 gene.
Pax6 - located on human chromosome
11, as we knew it plays a critical role in the development of the eye.
When a person has mutations in the pax6 gene
or some chromosome rearrangemnets caused a "position effect" on its normal
expression, it causes the eye malformation called Aniridia, loss of the iris of the
eye.
This feature can be an almost complete absence of the iris, or a
enlargement and irregularity of the pupil, or small slit-like defects not easy to be seen;
the effect on vision is variable too, from nearly normal to severe reduction in visual
acuity.
About half of the cases develop glaucoma which causes severe ocular pain
and can destroy residual vision.
Pax7 is located on human chromosome 1 and may be expressed in the developing nervous system and muscular systems.
Pax8 is on human chromosome 2, it
transactives two thyroid-specific genes, TG (thyroglobulin and TPO (thyroperoxidase).
Four splicing variants of pax8 (pax8a, 8b, 8c
and 8d) with distinct C-termini are expressed in human
kidney cell lines. Some pax8 isoforms also have been
found in thyroid, kidney and Wilms' tumors.
Pax9 - located on human chromosome
14, it is highly homologous to pax1 and may be
functioning in the development of the vertebral column.
Now you have seen a quite detailed picture of the model. - how genes control the body plan, and why genetic engineering has the magic power to modify the body shape and feature of the animal.
In next chapter, we will start to enter the genome kingdom of humans.
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