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Excample of an output of transcriptional profiling study using Illumina
sequencing performed in our lab. Shown is just a tiny fragment of the complete
list, copmprising about 7K genes revealing differential expression in the studied
mutant.
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Excample of an output of transcriptional profiling study using Illumina
sequencing performed in our lab. Shown is just a tiny fragment of the complete
list, copmprising about 7K genes revealing differential expression in the studied
mutant.
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One of such recent and very useful tools is Gorilla software, freely available at
http://cbl-gorilla.cs.technion.ac.il/.
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According to experimental evidence for the system under study, the hormone IAA, the
peptide TDIF, and the microRNA MIR165/6 are able to move among the cells. In the case
of TDIF and MIR165/6, the mobility is defined as diffusion and is given by the following
equation:
g(t+1)T[i]= H(g(t)[i]+ D (g(t)[i+1]+g(t)[i-1] – N(g(t)[i]))-b) (2),
where g(t)T[i] is the total amount of TDIF or MIR165 in cell (i). D is a parameter that
determines the proportion of g that can move from any cell to neighboring ones and is
correlated to the diffusion rate of g. b is a constant corresponding to a degradation term.
H is a step function that converts the continuous values of g into a discrete variable that
may attain values of 0, 1 or 2. N stands for the number of neighbors in each cell.
Boundary conditions are zero-flux. In the case of IAA, the mobility is defined as active
transport dependent on the radial localization of the PIN efflux transporters and is
defined by the equation:
iaa(t+1)T[i]=Hiaa(iaa(t)[i]+Diaa(pin(t)[i+1])(iaa(t)[i+1])+Diaa(pin(t)[i-1])(iaa(t)[i-
1])−N(Diaa)(pin(t)[i])(iaa(t)[i])−biaa) (3),
where Diaa is a parameter that determines the proportion of IAA that can be
transported among cells. The transport depends on the presence of IAA and PIN in the
cells and biaa corresponds to a degradation term. As in equation 2, H is a step function
21
that converts the continuous values to discrete ones and N stands for the number
of neighbors in each cell. Boundary conditions for IAA motion are also zero-flux.
21
The proposed model considers data that we identified and evaluated through an
extensive search (up to January 2012). It takes into account molecular interactions,
hormonal and expression patterns, and cell-to-cell communication processes that have
been reported to affect vascular patterning in the bundles of Arabidopsis. The model
components and interactions are graphically presented in the figure above. In the
network model, nodes stand for molecular elements regulating one another’s activities.
Most of the nodes can take only 1 or 0 values (light gray nodes in the figure),
corresponding to “present” or “not present,” respectively. Since the formation of
gradients of hormones and diffusible elements may have important consequences in
pattern formation, mobile elements TDIF and MIR, as well as members of the CK and
IAA signaling systems, can take 0, 1 or 2 values (dark gray nodes in the figure above)
Benitez and Hejatko, submitted.
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In comparison to the model shown on slide 21, the final version of the model
contains the predicted interactions (dashed lines).
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The initial conditions specify the initial state of some of the network elements
(figure above) and are the following :
I) In the procambial position (central compartment), CK is initially available and
there is an initial and sustained IAA input or self-upregulation. This condition is
supported by several lines of evidence. Also HB8, a marker of early vascular
development that has been found in preprocambial cells, is assumed to be
initially present at this position. These conditions are not fixed, however. After
the initial configuration, all the members of the CK and IAA signaling pathways,
as well as HB8, can change their states according to the logical rules.
II) In the xylem and phloem positions, it is assumed that no element is initially
active except for the CK signaling pathway and TDIF, both in the phloem
position.The level of expression for a given node is represented by a discrete
variable g and its value at a time t+1 depends on the state of other components
of the network (g1, g2, ..., gN) at a previous time unit. The state of every gene g
therefore changes according to:
gn(t+1)=Fn(gn1(t),gn2(t),..., gnk(t)) (1).
In this equation, gn1, gn2,…, gnk are the regulators of gene gn and Fn is a
discrete function known as a logical rule (logical rules are grounded in available
26
experimental data, for example see slide 20). Given the logical rules, it is possible
to follow the dynamics of the network for any given initial configuration of the
nodes expression state. One of the most important traits of dynamic models is the
existence of steady states in which the entire network enters into a selfsustained
configuration of the nodes state. It is thought that in developmental systems such
self-sustained states correspond to particular cell types.
According to experimental evidence for the system under study, the hormone IAA,
the peptide TDIF, and the microRNA MIR165/6 are able to move among the cells.
In the case of TDIF and MIR165/6, the mobility is defined as diffusion and is given
by the following equation:
g(t+1)T[i]= H(g(t)[i]+ D (g(t)[i+1]+g(t)[i-1] – N(g(t)[i]))-b) (2),
where g(t)T[i] is the total amount of TDIF or MIR165 in cell (i). D is a parameter
that determines the proportion of g that can move from any cell to neighboring
ones and is correlated to the diffusion rate of g. b is a constant corresponding to a
degradation term. H is a step function that converts the continuous values of g
into a discrete variable that may attain values of 0, 1 or 2. N stands for the number
of neighbors in each cell. Boundary conditions are zero-flux. In the case of IAA, the
mobility is defined as active transport dependent on the radial localization of the
PIN efflux transporters and is defined by the equation:
iaa(t+1)T[i]=Hiaa(iaa(t)[i]+Diaa(pin(t)[i+1])(iaa(t)[i+1])+Diaa(pin(t)[i-1])(iaa(t)[i-
1])−N(Diaa)(pin(t)[i])(iaa(t)[i])−biaa) (3),
where Diaa is a parameter that determines the proportion of IAA that can be
transported among cells. The transport depends on the presence of IAA and PIN
in the cells and biaa corresponds to a degradation term. As in equation 2, H is a
step function that converts the continuous values to discrete ones and N stands
for the number of neighbors in each cell. Boundary conditions for IAA motion are
also zero-flux.
Using the logical rules, equations 1–3, and a broad range of parameter values (not
shown here), it is possible fully to reproduce the results and analyses reported in
the following sections (see the figure above for the simulation time course).
26
The initial conditions specify the initial state of some of the network elements
(figure above) and are the following :
I) In the procambial position (central compartment), CK is initially available and
there is an initial and sustained IAA input or self-upregulation. This condition is
supported by several lines of evidence. Also HB8, a marker of early vascular
development that has been found in preprocambial cells, is assumed to be
initially present at this position. These conditions are not fixed, however. After
the initial configuration, all the members of the CK and IAA signaling pathways,
as well as HB8, can change their states according to the logical rules.
II) In the xylem and phloem positions, it is assumed that no element is initially
active except for the CK signaling pathway and TDIF, both in the phloem
position.The level of expression for a given node is represented by a discrete
variable g and its value at a time t+1 depends on the state of other components
of the network (g1, g2, ..., gN) at a previous time unit. The state of every gene g
therefore changes according to:
gn(t+1)=Fn(gn1(t),gn2(t),..., gnk(t)) (1).
In this equation, gn1, gn2,…, gnk are the regulators of gene gn and Fn is a
discrete function known as a logical rule (logical rules are grounded in available
27
experimental data, for example see slide 20). Given the logical rules, it is possible
to follow the dynamics of the network for any given initial configuration of the
nodes expression state. One of the most important traits of dynamic models is the
existence of steady states in which the entire network enters into a selfsustained
configuration of the nodes state. It is thought that in developmental systems such
self-sustained states correspond to particular cell types.
According to experimental evidence for the system under study, the hormone IAA,
the peptide TDIF, and the microRNA MIR165/6 are able to move among the cells.
In the case of TDIF and MIR165/6, the mobility is defined as diffusion and is given
by the following equation:
g(t+1)T[i]= H(g(t)[i]+ D (g(t)[i+1]+g(t)[i-1] – N(g(t)[i]))-b) (2),
where g(t)T[i] is the total amount of TDIF or MIR165 in cell (i). D is a parameter
that determines the proportion of g that can move from any cell to neighboring
ones and is correlated to the diffusion rate of g. b is a constant corresponding to a
degradation term. H is a step function that converts the continuous values of g
into a discrete variable that may attain values of 0, 1 or 2. N stands for the number
of neighbors in each cell. Boundary conditions are zero-flux. In the case of IAA, the
mobility is defined as active transport dependent on the radial localization of the
PIN efflux transporters and is defined by the equation:
iaa(t+1)T[i]=Hiaa(iaa(t)[i]+Diaa(pin(t)[i+1])(iaa(t)[i+1])+Diaa(pin(t)[i-1])(iaa(t)[i-
1])−N(Diaa)(pin(t)[i])(iaa(t)[i])−biaa) (3),
where Diaa is a parameter that determines the proportion of IAA that can be
transported among cells. The transport depends on the presence of IAA and PIN
in the cells and biaa corresponds to a degradation term. As in equation 2, H is a
step function that converts the continuous values to discrete ones and N stands
for the number of neighbors in each cell. Boundary conditions for IAA motion are
also zero-flux.
Using the logical rules, equations 1–3, and a broad range of parameter values (not
shown here), it is possible fully to reproduce the results and analyses reported in
the following sections (see the figure above for the simulation time course).
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More info about mouse at
http://www.informatics.jax.org/greenbook/index.shtml.
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Individula ICM cells of the embryo could be isolated and later re-introgressed
into the new embryo. These ICM cells are called embryonic stem (ES) cells. It is
very important technique that allows production of transgenic mice.
The isolated ES cells are transformed via foreign DNA construct and it is injected
within the embryo. The transformed cell becomes a part of the embryo and
might result into formation of different tissue types, among them the
spermatogonia or oogonia. i.e. the tissue that provides progenitor for sperm or
egg cells in the resulting chimera. Thus, the progeny of those chimeras will
inherit the modified cell with certain probability and these individuals will carry
the transgene in every cell of their body. Thus, the trangenic mice will be
produced.
This is very important mainly with regard of the knockout mutant (K.O.)
production. In the modified ES, the genes might be specifically eliminated via
DNA recombination. In that way, function of many of the mice genes was
identified.
E.g. the gene NODAL is expressed in the anterior portion of the primitive streak
that is equivalent to the Hensen’s node. nodal/nodal embryos are lethal, they do
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not undergo gastrulation and from almost no mesoderm.
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