Download Morphogens in biological development: Drosophila example

LSM5194
Morphogens in biological
development: Drosophila example
Lecture 29
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The concept of morphogen gradients
The concept of morphogens was proposed
by L. Wolpert as a part of the positional
information theory in 1969.
Morphogen gradients can be very shallow
and very sharp. In Xenopus, gradient of
activin can be formed experimentally over
100 um in 1 hour.
Teleman et al., Cell v 105, p 559, 2001
Several
mechanisms
have
been
proposed for the propagation of
morphogens through the tissue:
• diffusion
• transcytosis – endocytic relay from
cell to cell
Tabata, Nat Rev Gen v 2, p 620, 2001
• cell-cell contact by cytonemes
The main problem of morphogenesis can be formulated as one question.
How do cells know what is their developmental fate? Early in the history of
developmental biology it has become clear that for the cells to make a
decision on choosing their future, they need to know their position in the
developing tissue. This task to provide positional information to the cells was
ascribed to the morphogens – diffusible substances able to form gradients in
the tissue to enable cells to “read” both direction and the distance from the
organizing centers. As opposed to Turing’s idea, these morphogens do not
have to form any complex patterns themselves, only a system of long and
short gradients whose interpretation by individual cells will eventually result in
gradual creation of a complex pattern through the process of iterative
refinement. In this lecture we consider an example of a very well studied
developmental system – Drosophila embryo – which clearly demonstrates
how such patterning does occur in nature.
Despite earlier expectations, confirmed morphogens are almost all proteins
and not low weight molecules. These are proteins from TGFb, hedgehog and
wingless families. Interestingly most of morphogens require preprocessing,
such as proteolytic cleavage, to become active. The pro-protein and active
forms may also have very different life times ability to diffuse.
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The master plan of the Drosophila embryo
The creation of the body plan of
Drosophila by the gradient forming
proteins is perhaps the best understood
morphogenetic process.
Formation of morphogenetic fields starts
on the acellular level of syncytium.
Unless specified otherwise, the illustrations are from
Wolpert et al, Principles of development. 2002
The development of Drosophila is peculiar in a sense that during some 13
first cell divisions the cell-cell boundaries are not formed and the nuclei divide
in one giant cell called syncytium. Only after the newly formed nuclei
densely populate the near surface, cortex part of the syncytium the cellular
membranes are formed to form one layer of cells that covers the original
oocyte as a shell. The formation of pattern defining future body plan starts
long before this cellularization process begins. Therefore, in case of early
Drosophila development, the morphogen gradients are technically
intracellular.
The formation of the body plan is governed by five groups of genes which act
in a strict sequence and order. The emergence of pattern is mediated by their
complex logic of mutual activation and inhibition.
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Maternal genes define the body axes
Both anterior-posterior and dorsal-ventral axes
are defined by deposition of maternal mRNA
which is transcribed after fertilization. The
anterior end is defined by gene bicoid while the
anterior end is defined by a group of 9 genes
including nanos and caudal (red image below).
For establishment of the gross axis alignment, Drosophila embryo relies on
maternal genes. They are deposited as highly compact and highly localized
stores of RNA which begin to be translated into protein shortly after
fertilization and long before zygotic transcription begins. This maternal control
is exerted by at least 50 different genes while from the theoretical viewpoint it
would suffice only two genes to define the two axes. This proves highly
redundant and thus robust character of the development.
The anterior end of the embryo is defined by gene bicoid while posterior is
defined by a group of genes with main genes being caudal and nanos. Bicoid
as a transcription factor and acts directly as a morphogen by regulating
downstream gap genes. Nanos, on the other hand exists to establish a
gradient of another important gene – hunchback, as it suppresses its
translation in the posterior end.
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Dorso-ventral axis uses different mechanism
Specification of dorso-ventral axis occurs through activation of maternally deposited gene
dorsal by means of extracellular ligand spatzle deposited on the ventral part of the egg
shell through activation of Toll signaling pathway (analogue of the NF-kB pathway).
Very different from the previous slide mechanism is used to specify dorsoventral axis. In this case the mRNA for the gene dorsal (NF-kB) is deposited
homogeneously throughout the syncytium cortex. However, the
inhomogeneity is achieved through spatially heterogeneous action of a
signaling pathway which is a homolog of vertebrate NF-kB pathway. The
ligand, spatzle, is deposited outside of the egg on the internal surface of the
so-called viteline membrane that lines the inner surface of the egg shell.
The ligand is deposited on the ventral surface and therefore the pathway is
activated only there.
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Zygotic genes do actual segmentation work
Upon establishment of the gradient of dorsal, the rest of
the dorso-ventral pattern consisting of the 6 segments is
done by a group of 7 proteins that pattern ventral side
(rhomboid, twist and snail) separately from dorsal
(dpp, tolloid and zernknullt). The final result is
achieved through a complex activation-inhibition pattern
of gene expression.
Once the initial symmetry breaking gradients are established throughout the
syncytium, the more detailed patterning begins. This process is however
already defined by zygotically transcribed genes. The dorso-ventral pattern of
six segments is a typical example. In the ventral part, the established gradient
of dorsal activates twist, rhomboid and snail. The three genes are related by
complex pattern of activation and inhibition which results in spatio-temporal
pattern of gene expression which is eventually responsible for definition of
cell fates. On the dorsal side, the show is run by the gene product of
decapentaplegic or simply “dpp”. This is a homolog of BMP4, one of the
most important morphogens in the TGFb family. Its gradient is created by
complex interaction with other proteins, for example sog (short gastrulation)
which we will encounter in the next lecture again.
Note that this part of the embryo patterning occurs already on the cellular
phase.
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Gap genes pattern antero-posterior axis
After establishment of the antero-posterior gradient of
maternal genes, the zygotic gap genes switch on.
Hunchback is directly induced by bicoid while giant,
kruppel and knirps are induced downstream of
hunchback. All gap genes are transcription factors
necessary for the following tissue patterning. The shown
spatial patterns of expression result from complex pattern
of mutual activation and inhibition.
While on the stage of syncytium, the gap genes are induced by the maternal
genes whose gradients have already been established. Bicoid directly
induces hunchback. As shown on the slide there is low critical concentration
of bicoid below which hunchback expression is not activated. The location of
this threshold in the embryo defines the position of sharp hunchback
boundary. Multistriped patterns of giant, kruppel and knirps are results of
similar transformation of smooth gradients into binary outcome by use of
thresholds. As shown on the slide, the stripe of kruppel forms on the decaying
gradient of hunchback under the existence of two thresholds – for activation
and inhibition.
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Segmentation and pair-rule genes
Shortly after the gap genes, pair-rule genes eve and fushi
tarazu create 14 stripes of alternating expression which
will become after some modification the segments of the
larval body. Each strip has its own genetic control and is
not a result of a periodic process!
Emergence of periodic stripes of expression of genes even skipped (eve) and
fushi tarazu is a magnificent pattern formation phenomenon. Various
hypothesis and models were proposed to explain periodic stripes. To the
great surprise of both experimentalists and theorists, each stripe is
individually controlled by a specific combination of activation and inhibition by
the gap genes. Not surprisingly, the regulation regions of eve gene are the
most explored and the best understood gene control units up to date. Shown
here as an example is the anatomy of the second stripe of eve with the
corresponding details of the regulatory element on the eve gene. Overall, the
expression of eve (in this stripe) is activated by bicoid and hunchback while
giant and kruppel inhibit it. The group of pair-rule genes includes already
known to you gene hairy.
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Segment polarity genes finalize segmentation
Segment polarity genes finally mark the boundaries of the
body segments and provides them with antero-posterior
direction. The genes of this group code for such important
morphogens like wingless, engrailed and hedgehog and
they are connected by complex interaction network.
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Selector and homeotic genes define future of segments
Finally, the future of the Drosophila segments is
defined by homeotic and selector genes which are
homologous to the Hox genes of vertebrates.
These genes can change the whole organ into
another
organ.
For
example,
mutation
antennapedia results in substitution of antennae
by legs. The relationship to simple gradients can
hardly be followed on this level of development.
The complexity on this level exceeds the scope of this lecture. Just remember
that these genes define which segments will turn into which parts of a body.
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What to take home
• Morphogenesis in real biological systems is controlled by complex networks of
morphogens
• Each of them forms only simple gradient, yet the combination of these simple prepatterns being superimposed on each other can define layout of any complexity
• The relative importance of morphogen gradients diminishes with the size of embryo
and with the level of development
• To achieve necessary robustness and scale independence, morphogenetic
networks must possess high redundancy and non-linearity achieved through multiple
positive and negative feedback loops
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