.

WEL COME
SEMINAR-II

ON

Somashekhar Guddadamath
PGS07 AGR4586
The regulatory apparatus- 2 complementary components.

i) Transcription factors- specific sequences in the DNA,

ii) signaling molecules- communication between the cells

Every gene contains regulatory sequences that control when

and where it is expressed.

The regulatory sequences are arranged in units that are

termed cis-regulatory modules (CRM’s). Every cis-regulatory
module contains a cluster of different transcription factor
binding sites.
Specification is the process by which cells acquire identities,
that means the process by which cells reach the specific
regulatory state that defines their identity and the
differentiation genes that they express.

The transcription factors together with signaling cues from

the neighboring cells activates a number of cis-regulatory
modules (CRM’s) . The active modules turn on the expression
of regulatory genes that construct the next regulatory state of
the cell until specification and differentiation are achieved.
A node in the network is a regulatory gene and its multiple
cis-regulatory modules that receive input from elsewhere in
the network and provide output that is destined to targets
elsewhere in the network. (Fig )

Studying regulatory networks enables the understanding

of the mechanism underlying the developmental process at
the most fundamental level.
• Genome sequencing enables the application of comparative
computational tools that help detect regulatory genes and
their cis regulatory modules.

• Gene regulatory networks for development look diverse in

both structure and function.

• There are number of gene regulatory network models that

describe domain specification of various organisms, some of
the gene regulatory network models are….
 Some models of gene regulatory networks for specification and
differentiation

Organism Domain specification

Sea urchin (Sp) Endo mesoderm
Starfish (Am) Endoderm
Mouse (Mm) Pancreatic β-cells
Mammals B-cell specification
Vertebrates Heart field specification
Frog (Xl) Mesoderm
Ascidian (Ci) Notochord
Fruit fly (Dm) Dorsal-Ventral axis
Fruit fly (Dm) Heart field specification
Nematode (Ce) Vulva
Am, Asterina miniata, Ce, Caenorhabditis elegans, Ci, Ciona intestinalis,
Dm,Drosophila melanogaster, Mm, Mus musculus, Sp, Strongylocentrotus purpuratus,
Xl, Xenopuslaevis
• General features that are universal to all developmental
networks.

The specific combination of transcription factors --

leads to activation or repression of a particular cis-regulatory
module, not just the action of a single gene.

The networks are modular and can be resolved into

sub circuits in which every sub circuit is responsible for a
specific developmental task. Different sub circuits are active
in different domains and times in the embryo.
 The network sub circuits are composed of typical
functional elements: signaling, specification establishment
and persistence by, for example, positive-feedback loops,
alternative fate exclusion and boundary formation by
repressors.
 Schematic diagrams of the sea urchin
(Strongylocentrotus purpuratus) embryo development.
Gene Regulatory network for endo mesoderm specification in the Sea urchin embryo
(Davidson et al. 2002)
Gene Regulatory network for endomesoderm specification in the
Sea urchin embryo

• All the interactions are encoded in DNA in the form of

cis-regulatory elements.

• In every step and domain only some of the network

nodes are active in a given cell, depending on its
regulatory state.

• Early stage: signaling initiate domain specification.

• Later stage: Specification state establishment and

persistence
Early stage:

Asymmetric distributions of particular proteins, mRNA,

or even cellular components in the egg, localization of
these components in the egg gives rise to localized
regulatory activity in the embryo, which leads to axis
formation and specification initiation.

In early cleavage in sea urchin embryo , activation of

particular regulatory genes in the micromeres that
turn on three primary specification sub circuits.
• The three sub circuits, named by the principal genes that
compose them are

1. β-catenin-wnt8-blimp1,

2. Pmar1-Repressor of the micromeres, and

3. delta-notch signaling.
1. Dynamic Spatial Patterning by β-catenin-wnt8-blimp1 Sub
circuit:

When β-catenin is stabilized so it enters the nucleus, it binds to

the transcription factor TCF1 and forms a permissive complex
that allows transcription.

In cells where β-catenin is degraded by GSK-3, Groucho binds

to TCF1 and together they form a dominant-repressive
complex.

Thus, stabilization of β-catenin leads to its entrance to the

nucleus, where it removes the repression that is induced by
Groucho so that cis-regulatory modules that have TCF1 binding
sites are activated.
 The β-catenin- TCF1 complex together with Blimp1 activates the
expression of Wnt8 signaling molecule.

Wnt8 signal is received by the neighboring cells and induces

further stabilization of β-catenin by activating the Disheveled protein.

Wnt8 signaling has a positive feedback into Blimp1 and Wnt8 in

the same ring of cells in a community effect and also turns on this sub
circuit in the next tier of cells.
 Blimp1 auto represses itself, and because its input is
required for the activation of wnt8, wnt8 expression is also
stopped.

The expression of these two genes and of other

particular genes regulated by β-catenin-TCF1 forms a ring.

The use of one of the sub circuit internal components to

sequentially shut it off is a elegant way to control well-timed
spatial expression pattern.
 The cis regulatory module that controls the early
expression of wnt8 has both Blimp1 and TCF1 binding sites,
and both inputs are necessary for the activation of the gene.

TCF1 binding sites are also responsible for the

repression of the wnt8 gene outside the vegetal plate and for
the spatial localization of this sub circuit.
β-catenin-wnt8-blimp1 Sub circuit:
 The Disheveled protein is distributed asymmetrically
in the egg and is localized in the future vegetal pole
 Nuclear localized β-catenin binds to TCF1 and removes the
repression induced by the globally expressed Groucho
2. Autonomous Micromere Specification by Pmar1
Sub circuit:
In the micromeres, nuclearized β-catenin together with the
maternal transcription factor Otx activates the expression
of a repressor, Pmar1.

Pmar1 represses a ubiquitous repressor, Repressor of

micromeres, allowing transcription of micromere-specific
genes such as the signaling molecule Delta and the
transcription factors Ets1, Alx1, and Tbr
Pmar1-repressor of micromere sub circuit.
3. SMC Specification Induced by delta-notch Signaling

One of the regulatory genes downstream of Pmar1 regulation

is the signaling molecule Delta which binds to Notch receptor
in the neighboring tier of cells.

As a result the Notch intracellular domain (NIC) enters the cell

nucleus and competes with the co repressor, Groucho, on
binding to the transcription factor Suppressor of Hairless
[Su(H)]

NIC and Su(H) form an activating complex so the repression

that was induced by the corepressor is removed. The Su(H)-
NIC complex activates the expression of the gene gcm, which
initiates the pigment cell specification state establishment.
SMC Specification Induced by delta-notch Signaling

Pmar1 repression
 LATER STAGE:

SPECIFICATION STATE ESTABLISHMENT AND PERSISTENCE

• The later stage is governed by positive-feedback loops that

promote the establishment of specification states and by
turn-on of localized repressors.

• The sub circuits involved in the specification of two

mesoderm lineages and the endoderm lineage are discussed
here.
Early Mesoderm Sub circuits: PMC and SMC Specification

The mesoderm territory consists of the cells between the

outer cell territory, the ectoderm, and the inner cell territory,
the endoderm.

two mesoderm cell lineages-

-the skeletogenic lineage and

-the pigment cell lineage

The skeletogenic lineage is formed from the large micromere
descendants, the PMCs.

In the skeletogenic lineage, Ets1, Alx1, and other regulatory

genes activate a battery of skeletogenic differentiation genes,
e.g., bio mineralization proteins (sm27 and sm50),
glycoproteins (msp130 and msp130L), and cyclophilin and
ficolin. (fig)
The pigment cell lineage is formed from the SMCs, the
descendants of the veg2 ring of cells that are the immediate
neighbors of the PMCs .

In the pigment cell lineage Gcm drives the expression of the

differentiation gene battery, which encodes pigment synthesis
enzymes such as
SuTx (sulfotransferase), Dpt (dopachrome tautomerase),
Pks (polyketide synthetase), and
FvMo (flavinecontaining monooxygenase) (fig)
• gcm expression continues long after the Delta-Notch signal
turns off. The expression of this key specification gene is
maintained by a simple positive-feedback loop of the gcm
onto itself.

• A positive-feedback loop is a typical functional element that

gene regulatory networks use to stabilize and lock cells into a
specification state.
• Another feature of this stage is a local exclusion of alternative
fate, i.e., local repression of genes that do not belong to the
specified domain. Gcm contributes to this function by down
regulating the expression of the PMC transcription factor,
alx1, in the SMC

Pmar1 repression
• when gcm expression is turned on specifically in the PMCs
they cease the expression of some skeletogenic genes and
acquire pigment cell properties and vice versa true.

• a single gene, in this case gcm, is sufficient to change the PMC

into SMC demonstrates how crucial exclusion control is for
the normal development of the embryo.
Mesoderm and Endoderm Specification Sub circuits: veg2
Subdivision into SMC and Gut

The endoderm territory in the sea urchin larva constitutes the

gut, the veg2 ring of cells form two rings of cells,
-an inner ring of cells
-an outer ring of cells

The inner ring of cells continues to receive the Delta-Notch

signal from the micromeres and eventually becomes the SMC.

The descendants of the outer ring of cells form most of the

endoderm.
PMC Delta activates gcm in the SMC. However, the Delta-
receiving endoderm cells express foxa, which down regulates
gcm expression.

β-catenin-Blimp1-wnt8 sub circuit is active in the future

endoderm territory.(18 h after fertilization). (fig)

Blimp1 activates a later isoform of the otx gene, otxβ. Otxβ

positively regulates itself and gatae expression
• GataE activates further expression of otxβ, and a stabilizing
feedback loop is established between otxβ and gatae.

• Otxβ and GataE regulate the expression of essential

endoderm specification genes, bra and foxa. otxβ, gatae,
foxa, and bra control the endoderm specification and
differentiation and are necessary for gastrulation.
The key DNA components that control the execution of this sub
circuit are the cis regulatory modules that contains binding sites
of Blimp1, GataE, and Otxβ (fig)

Interfering with either the trans input, by blocking the

translation of a transcription factor, by mutating the relevant
binding sites, results in the similar decrease of expression.

These results show that the GataE, Otxβ, and Blimp1 inputs are
necessary for Otxβ expression in the endoderm.
A cis-regulatory module that controls the expression
of the otxβ in the endoderm
The expression of otxβ transcript
 Requirement of SpOtx in Cell Fate Decisions in the
Sea Urchin Embryo
Xiaotao Li et al. 1999
• MATERIALS AND METHODS
Plasmid constructions.
• The plasmid encoding the SpOtx fusion protein was generated by fusing, a
fragment containing the N-terminal 296 amino acids of Drosophila Engrailed with
the SpOtx homeodomain.

• Two plasmids were constructed one contained the wild-type SpOtx

homeodomain [Eng– OtxHD(K)] and the other had the lysine at position 50
replaced with a glutamine [Eng–OtxHD(Q)]

• Replacing the lysine with glutamine essentially eliminates the ability of SpOtx to
bind to its TAATCC/T target site

• An SpOtx cDNA was used as a polymerase chain reaction (PCR) template to

generate the fragment containing the SpOtx homeodomain.
• Embryo culturing and microinjection

S. purpuratus were obtained from Marinus (Long Beach, CA). Gametes

were collected and fertilized, and embryos cultured (Mao et al., 1996).
• Messenger RNA synthesized from , Eng-OtxHD(K), or Eng-OtxHD(Q)
constructs was injected at about 0.05 pg per egg.

• CAT reporter gene analysis,

linearized Spec2a-CAT, Endo16-CAT, or pCAT1 DNA was injected together

with pCS2-ENG-N, Eng-OtxHD(K), or Eng-OtxHD(Q) mRNA at
approximately 2000– 4000 copies per egg
• RESULTS AND DISCUSSION

• Dominant Repression of SpOtx Target Genes.

• SpOtx(a) and SpOtx(b) contain potent transcriptional activation
domains in their N-terminal and C-terminal regions and are
implicated in the activation of several genes in early S.
purpuratus development,

• Unlike most other homeodomain-containing proteins, Otx

proteins bind with high affinity to a specific DNA element,
TAATCC/T, due to a lysine residue at position 50 of the
homeodomain.
• To determine whether the SpOtx fusion proteins would repress the
activity of transcriptional regulatory regions containing Otx sites,
they injected Eng– OtxHD(K) or Eng–OtxHD(Q) transcripts into S.
purpuratus eggs along with either Spec2a-CAT or Endo16-CAT reporter
genes.

• Embryos injected with Spec2a-CAT or Endo16-CAT had the expected

activity, whereas activity from both reporter constructs was severely
attenuated when Eng–OtxHD(K) RNA was coinjected.(fig)

• coinjection of Eng– OtxHD(Q) RNA had no effect on reporter gene activity

even though the same levels of Eng–OtxHD(K) or Eng–OtxHD(Q) RNAs
were present in the injected embryos.

• These results demonstrated that the Engrailed–SpOtx fusion protein

inhibited reporter gene activity and that a functional Otx homeodomain
was required for the inhibition.
• They tested the endogenous gene expression by expressing the
Engrailed–SpOtx fusion proteins, and monitored genes expressed
specifically in

aboral ectoderm (Spec1, Spec2a, CyIIIa actin),

endoderm (Endo16),

primary mesenchyme (SM50), and

secondary mesenchyme (SpBra) cell types using RT-PCR analysis

• Injection of Eng–OtxHD(K) RNA resulted in a substantial
reduction in Spec1, Spec2a, CyIIIa actin, and Endo16 expression
but had no significant effects on SM50 or SpBra expression.

• Injecting Eng–OtxHD(Q) did not alter the expression of any

endogenous genes that were tested. Primers specific for the
Eng–OtxHD RNAs showed that both transcripts were present at
similar levels in the injected embryos.
• These results showed that the Engrailed–SpOtx fusion protein
was able to specifically inhibit the expression of a subset of
genes. Spec1, Spec2a, and Endo16 all contained Otx elements
in their transcriptional control regions, and these genes were
directly repressed by the Engrailed–SpOtx protein.

• In contrast, Otx elements are not involved in CyIIIa actin

gene expression. This indicated that, at least for CyIIIa actin,
the Engrailed– SpOtx fusion protein was unlikely to act
directly to inhibit transcription.
• SpOtx(a) has a role in the sea urchin embryo associated with
the differentiation of aboral ectoderm and endoderm cell
types.

• a major role for SpOtx(b) is likely to be in the patterning of

structures along the fivefold symmetric axes of the radial
adult body
CONCLUSION