BBS1005 Human Genetics, Reproduction …. - 03/06/22
Case 7 - Week 5
The Hox genes are usually found in four clusters (A-D) and located
What does the embryo look like at week 5?
on different chromosomes, at 7p15, 17q21.2, 12q13, and 2q31. Each
cluster consists of 13 paralog groups with nine to eleven members
Week 5 of pregnancy :)
assigned on the basis of sequence similarity and relative position
within the cluster.
Quick recap
Hox genes are transcribed at different
Week 1: fertilization - cleavage - compaction - differentiation -
parts of the body, the Hox specificity
cavitation - hatching - implantation
probably already has been established
Week 2: Implantation - cavities
before the somites themselves are
Week 3: Gastrulation - Neurulation
actually made.
Week 4: body folds
For e.g. during the somitogenesis the boundary for Hox6
paralogs is already established to group at the
cervical/thoracic boundary, Hox10 falls at the thoracic/lumbar
Week 5 boundary, and Hox11 paralogs fall at the lumbar/sacral
At five weeks, the embryo is still very boundary; segment diversity is specified by a specific combinatorial code.
small but growing quickly. At this stage, the embryo is around 1.5 - According to this, the loss of a certain Hox gene or protein will
2 mm long. affect the combinatorial code and therefore significantly alters
The embryo’s outer layer of cells has developed a groove and has the vertebrae development in such a way that an anterior
formed the neural tube. The neural tube will now quickly form the homeotic transformation takes place; cranialization; the loss of a
brain and spinal cord. certain Hox gene or protein then results in the vertebrae
developing into a previous boundary, cranialising the vertebrae.
The nervous system is already developing, and the foundations for
its major organs are in place (due to the somites & body folds). Conversely, the gain of a certain Hox gene in vertebrae causes it
The heart is forming as a simple tube-like structure. The baby to develop into a vertebra, which is normally found more
already has some of its own blood vessels and blood begins to posteriorly. For e.g. Hox 6 then induces rib formation in the neck.
circulate. A string of these blood vessels connects to the mother The gain of a Hox gene, and therefore its
and will become the umbilical cord. transforming into a normally later (more
posterior) vertebrae may also be called
● The ectoderm is beginning to form the nervous system
caudalisation.
(including the brain and spinal cord from the neural tube). It
will also form the skin, hair, and nails
● The mesoderm is becoming the circulatory system with the
development of the heart and blood. It will also develop into
Hox genes form a sequential pattern over the anterior-posterior
the bones, muscles, and kidneys.
axis of the embryo. This sequential and functional patterning and
● The endoderm will eventually become the lungs, intestines,
differentiation occur due to Hox proteins being transcriptional
and liver.
factors, which thus in their turn activate or suppress certain genes
See resources->3D interaction
in the cell. As mentioned before, the latest Hox gene activated in a
cell determines its destination;
HOX gene ○ Hox5 causes the cervical vertebrae to form
○ Hox6 the thoracic vertebrae
What is the HOX gene and its function in embryo development? ○ Hox9 helps Hox6 to form the thoracic vertebrae
○ Hox10 causes the lumbar region to form
Homeobox (HOX) genes are a set of homeotic genes1, which
○ Hox11 causes the sacral segments to form.
control the body plan of an organism along the cranial-caudal
If a certain Hox gene does not activate on the right place, the
axis.
previous formation will be caused: cranialisation.
Every Hox protein, encoded by Hox genes, in itself does not induce
If a certain Hox gene is activated prior to healthy condition, a
certain specialization, but these proteins are transcriptional
normally later (more posterior) vertebrae is formed: caudalisation.
factors that will induce the protein necessary to form a certain
part of the body. The Hox gene sequence, in 5-6-9-10-11
fashion is found in the DNA along one part of
So Hox genes determine which limbs grow in each segment. the DNA of the third chromosome in a 3’-5’
way. -> The 3’ genes are expressed earlier
and more anteriorly than the 5’ genes.
1
Homeotic genes = transcription factor genes that form the body plan of an organism,
which most often are fairly equal over multiple species
, Notch signaling pathway
Hox genes can also affect other Hox genes, Hox1 for example
activates Hox2, where it is the same story for Hox4 on both Hox5 Delta is transcripted, translated and will then be expressed on the
and Hox7, and Hox7 causes Hox8 and Hox9 to be transcribed, surface of the cell. This is a Notch ligand and activates the Notch
probably as normal transcription factors. on the neighboring cell. The Notch will identify the Delta and
activate the Notch pathway.
Not all Hox genes only activate though,
some Hox genes suprress a previous Hox The activated Notch therefore releases Notch Intracellular Domain
protein by creating miRNA found in the DNA (NICD), which is transferred into the nucleus. The NICD then forms
sequence which interrupts with the previous a complex with the Rbpj mediator, which upregulates the
Hox proteins and therefore deactivates their downstream gene transcription of Hes7. This synchronisation
function. e.g. Hox4 on Hox1, Hox2, and Hox3 and like Hox7 onto Hox5. makes sure that all cells in a certain range will be equal, mainly
while they can be affected by mitosis and arrest their gene
The last Hox gene (Hox13B) also makes sure that somitogenesis is
transcription; altering its timing if Notch signalling would not
stopped by decreasing the Wnt3a and FGF production (both
occur.
explained later).
The Hes7 dimer suppresses the Hes7
Hox genes are regulated by Gap proteins and pair-rule genes, like
promotor. So hen Hes7 is transcripted into the
Bicoid, Hunchback, Giant and Krüppel in drosophila (mentioned in
mRNA, then translated into a protein, and
case 3). In the human embryo, the Hox specificity is established
accumulated in the cytoplasm, it will repress
during gastrulation and tail-bud stages. The Hox gene expression
its own transcription.
is regulated by Wnt, Bmp11, and retinold signalling.
The Hes7 dimer also suppresses the Delta
The different signaling pathways promotor, therefore it decreases the amount of Delta protein
made, decreasing the excitation of the neigbouring cell.
WNT*, FGF*, Notch, HES Less NICD in the neighbouring cell results in less Hes production,
which somewhat equalizes the synchronisation, but it also allows
Fgf signaling pathway more Delta to be formed.
Fibroblast growth factor (FGF) binds to
the FGF receptor and is helped by heparin
sulphate proteoglycan (Hgsp).
The binding of FGF to its receptor causes
the activation of Ras and the
FGF and Notch, both have
phosphorylation cascade. This cascade
an effect on the negative feedback loop of Hes7. there is also an
will then cause RAF, MRK, and ERK to be
influence of Wnt pathway, which will inhibit Notch pathway.
phosphorylated.
The phosphorylated ERK then moves to the nucleus, where it
regulates the gene expression.
Wnt signaling pathway
Wingless-related integration site (WNT) is
secreted and binds to the WNT receptor or
the so-called frizzled receptor.
In mammals, there are 19 WNT and 10
frizzled receptors.
The segmentation clock
The binding of WNT molecule to the
receptor starts the intracellular signaling
How does the segmentation clock work?
cascase that involves three pathways:
At the end of week 3, the body folding
1. Canonical WNT pathway starts. Somites start forming at day 17,
The binding of WNT leads to the signal transduction to DSH and by day 21, there are 3 somites.
and AXIN, which in turn prevents the degradation of
B-catenin. As its degradation is prevented, B-catenin
Somitogenesis
accumulates in the cytoplasm and then diffuses to the
In the 3rd week of development, just
nucleus, where it binds to the transcriptional compressors
after gastrulation, we have three germ layers: endoderm,
TCF/LEF. This binding consecutively results in new
mesoderm, and ectoderm.
transcription and WNT signaling.
2. Planar cell polarity pathway The mesoderm has three subdivisions:
3. Calcium-signaling pathway - lateral mesoderm (parietal and visceral) -> heart,
vessels body wall and limbs
WNT pathway normally inhibits the NOTCH pathway.
- intermediate mesoderm -> kidney and gonads
, - paraxial mesoderm -> head somites, skin connective 1. Bottom tier: single cell oscillators
tissue, muscles, cartilage The bottom tier consist out of single cell oscillators2, which
regulate the segmentation by negative feedback mechanisms of
Hes7. This happens in the tailbud.
There is an oscillating expression pattern of Hes7 mRNA and
protein. As mentioned before, the oscillatory expression of Hes7 is
regulated by negative feedback of itself; Hes7 gene transcription
forms Hes7 mRNA, which is translated into the Hes7 proteins.
From the paraxial mesoderm, the somites will be formed.
The Hes7 protein forms a dimer with another Hes7 protein by the
Somites are embryonical transitional organs that will give rise to
helix-loop-helix domain, wich binds onto the Hes7 promotor and
the axial skeleton, ribs, dermis of the skin, and striated muscles of
blocks its own synthesis.
the neck, trunk, and limbs.
In this way, accumulation of Hes7 will repress the Hes7 mRNA
Somitogenesis (day 17)is the development of
synthesis and will also cause its own degradation via
somites. This happens sequentially in an anterior to
uniquitination. Therefore the Hes7 levels drop and the Hes7
posterior way. The (posterior) part where the somites
promotor is no longer blocked; Hes7 transcription can be initiated
are not present yet, is called the pre-somitic
again.
mesoderm. At the end of the pre-somitic mesoderm,
Transcription of the Hes7 gene can thus only find
the tailbud, there are mesodermal progenitor cells
place when its levels are 0 or very low.
(MPCs) present.
On the bottom tier the Hes gradients are
Somitogenesis is dependent on the segmentation
established by this negative feedback in the
clock, which is the cyclic expression of specific genes. The cyclic
single cell oscillators; the levels of Hes therefore
genes include members of the NOTCH and WNT signaling
rise and fall and create wave fronts where
pathways that are expressed in an oscillating pattern in
increased Hes proteins establish the gradient
pre-somitic mesoderm.
differently over all cells.
Segmentation clock
2. Middle tier: local synchronisation
At the area of the wavefront, the separation of the
The middle tier consists out of the selfsustaining synchronization
mesoderm starts, so the segmentation into somites
between neighbouring cells. This takes place in the pre-somitic
takes place.
mesoderm.
The Hes genes generate travelling waves. These waves are
created over multiple cells and have to be synchronised with each
The cells of the presomitic mesoderm that are proliferating will
other in a horizontal fashion; it uses the oscillation of the cell level
express the factors FGF and Wnt. They will then push the already
on its own. However, while the cells are interconnected with each
proliferated cells cranially until one point, they won’t express FGF
other it is much more precise and also aligned with other cells.
and Wnt anymore. This is because the factors are degraded,
which will decrease the concentration. These is an interaction between delta and notch between these
This will form the gradient of FGF and Wnt that is present in the two neighboring cells. As mentioned before, the interaction
cells of the presomitic mesoderm. between delta and notch will activate the Notch signaling
pathway, which will then again activate the mRNA transcription of
So, in the presomitic mesoderm there are oscillations of the
the Hes7 gene. The Hes7 protein will not only suppress its own
concentrations of the factors. However, these oscillations will stop
synthesis, but also the expression of delta.
at the wavefront, because of the threshold of FGF and Wnt. This
arrest will induce the segmentation. When the Notch pathway is activated, NICD is released an travels
into the cell nucleus where it activates Hes7. However, the NICD
also activates Lfng, which inhibitis the NICD production.
Since the Hes7 inhibits itself and the Lfng, the Hes7 created by the
Notch pathway:
○ Inhibits itself
○ Partially decreases the inhibition of the Notch pathway
(since it decreases the Lfng, which inhibits this pathway).
The segmentation clock consists of
three main steps; three-tier model. The three tiers are the bottom On top of the Notch signalling, the FGF pathway phosphorylates
tier, middle tier, and upper tier. ERK and therefore creates the active pERK, which induces
expression of Dusp4 and Dusp6. The pERK also activates the Hes7
transcription, which (again) inhibits itself (just like all the other 100
times :) ).
2
Oscillators are proteins which have a negative feedback loop on the translation of its own mRNA (e.g.
Hes)
, The Hes7 now also inhibits the activation of Dusp4 by pERK; the The Mesp2 will also again inhibit the Notch signaling.
Dusp4 inhibits the pERK and therefore lowers the Hes7 production,
however not very extreme while the Hes 7 thus also inhibits the
Dusp4.
However, there is also the Wnt signalling itself, which will also
influence the feedback cycle of the oscillations. Wnt will break the
formation of a certain complex, which will then fall apart in APC,
The global picture of these circuitries show a pre-somatic
B-catenin, CKIa, Axin, and GSK3B.
mesoderm where on the posterior side new cells are generated
Normally, the target is to free B-cathenin, which is a transcription and the FGF and Wnt are produced and from there further
factor that can migrate to the nucleus to act on mRNA distributed. At the same time, the posterior side of the
transcription. However, in this case, Wnt signalling is important to pre-somatic mesoderm keeps proliferating: new cells are made
free GSK3B, which is a kinase. This kinase will inhibit the Notch and this side lengthens, retracting the FGF/Wnt production away
signalling pathway. Due to the Notch inhibition, less NICD is from the middle and creating a wave with a decreasing FGF/Wnt
produced, decreasing the Hes 7 transcription. concentration.
Also in this Wnt signalling pathway there is a feedback The waves created in this manner allows the anterior side of a
mechanism. When the complex is broken, the factor Axin will pre-somatic group of cells to produce higher levels of oscillators,
come free, which will inhibit the Wnt signalling immediately. So while further posterior of the pre-somite, the level of these
then the complex which fell apart, is again formed. oscillators are lower.
This means that when there is high Wnt signalling, there will be low At the determination front, the
Notch signalling. High Wnt signalling will result in high Axin. phases of the cells within a
While Axin is produced by the Wnt signalling, it inhibits the Wnt, pre-somite are established, which
and therefore will lead to an increase of the Notch signalling result in the anterior side of the
(since Wnt normally inhibits the Notch). pre-somite establishing high levels
At the final wave front, there no longer is a Wnt concentration, fully of Mesp2 and the posterior side
turning off the Wnt3a signalling, and therefore allowing the Notch lower Mesp2 and the oscillatorions
signalling to turn on. being arrested.
The turned off Notch signalling allows for EphA4 being produced,
while turned on Notch signalling causes eprhinB2 to be produced.
So on the anterior side of the somite, high EphA4 is created, and
on the posterior side, high ephrinB2 is produced.
Due to these altering concentrations of proteins in the cells found
within the somites, the embryo known how to split up the cells to
3. Upper tier: global control of slowing and arrest create separate somites.
The upper tier is the global control of slowing and
arrest.
At the wave front, arrest of the oscillations occur.
At the wave front, both FGF and Wnt cannot affect
the cell anymore, since there is almost no Wnt and
FGF. Therefore the cell will be totally dependent on
the Notch signalling on its own and the Hes7.
➢ Without FGF, no new pERK can be formed.
➢ Without Wnt, and thus GSK3B, only the break of the Notch
will be released a bit.
-
the Notch signalling now allows the Mesoderm Posterior 2 (Mesp2)
concentration gradients in the cells to increase. the Mesp2 can
only be formed when the Wnt and FGF signalling is turned off.
