Gene Regulation during Development 200431060022 陈国荣

Gene Regulation during

Development

200431060022 陈国荣

Outline

Part One: Three Strategies by which Cells Are instructed to express Specific Sets of Genes during Development

Part Two: Examples of the Three Strategies

Part Three: The Molecular Biology of Drosophila Embryogenesis

Part One: Three Strategies for Specific Genes Expression

Some mRNAs Become Localized within Eggs and Embryos due to Intrinsic Polarity in the Cytoskeleton

Cell-to-Cell Contact and Secreted Cell Signaling Molecules both Elicit Changes in Gene Expression in Neighboring Cells

Gradients of Secreted signaling Molecules Can Instruct Cells to Follow Different Pathways of Development based on Their Location

mRNA Localization

mRNAs serve as a critical regulatory molecule

They are transported along elements of the cytoskeleton which have intrinsic polarity

The transportation is realized by an “adapter protein”

“Adapter” proteinsContaining two

domainsOne recognizing the

3’ UTR of the mRNAThe other associates

with the components of the cytoskeleton

Thereby it crawls along the actin filament

Cell to Cell Contact and Secreted Cell Signaling Molecules• Three steps:

– The synthesized signaling molecules are deposited in the membrane or secreted into the extracellular matrix.

– They are recognized by the receptor on the surface of recipient cells

– The changes in gene expression in the recipient cell is achieved through the signal transduction pathways

The Signal Transduction Pathways

Ligand-receptor interaction induces kinase cascade that modifies regulatory proteins present in nucleus.

Activated receptor cause the release of DNA-binding protein so it can enter the nucleus, regulate gene transcription.

The intracytoplasmic domain of the activated receptor is cleaved to enter the nucleus and interact with DNA-binding protein.

Gradients of Secreted Signaling Molecules Influences Development through Cells’ LocationThe influence of location

on development is called positional information

Signaling molecules that control position information are sometimes called morphogens

The morphogens are distributed in extracellular gradient

Part Two: Examples of the Three Strategies

The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO gene

A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo

Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium, B. subtilis

A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect CNS

A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube

No.1 The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO gene After budding to

produce a daughter ,a mother cell can switch mating type

The switching is controlled by the product of the HO gene

The HO gene is activated in the mother cell but kept silent in the daughter cell

No.1 The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO gene

Ash1 mRNA is localized during budding

The mRNAs are transcribed into repressor which represses the transcription of HO gene in the daughter cell

Thereby mating type is controlled

Ash1 mRNA Distribution

No.2 A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo

Macho-1 mRNA is initially distributed throughout the cytoplasm of unfertilized eggs

Localized to the vegetal(bottom)region shortly after fertilization,

Ultimately inherited by two cells of the eight-cell embryos,

Thus the two cells go on to form the tail muscles

Macho-1 regulatory protein

Macho-1 regulatory protein is a major determinant to form muscle .

The Macho-1 mRNA encodes a zinc finger DNA-binding protein that is believed to activate the transcription of muscle-specific genes, such as actin and myosin.

Macho-1 is made only in muscles cells.

NO. 3 Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium, B. subtilis

The septum produces two cells remain attached through abutting membranes.

The smaller cell, called forespore, ultimately forms the spore.

The larger cell ,the mother cell, aids the development of the spore.

The forespore influences the expression of genes in the neighboring mother cell.

Relationship between the two cells:

NO. 3 Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium, B. subtilis

Steps for influences:I. The active form σF in forespore activates spoIIR

gene

II. The product spoIIR is secreted into the space between the mother and the daughter’s membranes

III. SpoIIR triggers the activation of the σE in the mother cell through proteolytic process

IV. Activated σE initiates transcription of the target genes

Asymmetric gene activity in the mother cell and forespore in the B.subtilis

No.4 A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect CNS Neurogenic ectoderm is a

sheet of cells that will develop into nerve cord in insect embryos

It can be divided into two cell populations: One group remains on the

surface of the embryo and forms epidermis

The other moves inside the embryo to form the neurons of the ventral nerve cord

The developing neurons contain a signaling molecule on their surface called Delta

The Delta’s receptor called Notch is on the skin cells’ surface

No.4 A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect CNS

I. Notch receptor is activated by DeltaII. The intracytoplasmic domain of Notch is

released to enter nuclei and associate with Su(H)

III. Su(H)-NotchIC complex activates genes that encode transcriptional repressors which block the development of neurons

Steps for the Skin-Nerve Regulatory Switch:

No.4 A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect CNS• Notch signaling does not

cause a simple induction of the Su(H)– In the absence of signaling,

Su(H) is bound to repressor proteins including Hairless, CtBP, and Groucho

– When NotchIC enters nucleus, it displaces these repressor proteins

– Su(H) now activates the very same genes that it formerly repressed

No.5 A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube

In all vertebrate embryos, there is a stage when cells located along the dorsal ectoderm move toward internal regions of the embryo and form the neural tube.

Cells located in the ventralmost region of the neutral tube form floorplate, where the secreted signaling molecule Sonic Hedgehog(Shh) is expressed. Shh functions as a gradient morphogen.

No.5 A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural TubeShh diffuses through the extracellular

matrix of the neutral tube and forms a gradient.

The graded distribution of the Shh protein leads to the formation of distinct neuronal cell types in the ventral half of the neural tube.

High and intermediate levels lead to the development of the V3 neurons and motorneurons,respectively.Low and lower levels lead to the development of the V2 and V1 interneurons.



The activation of the Shh receptor allows a previously inactive form of Gli transcription activator to enter the nucleus in an activated form.

The gradient of Shh leads to a corresponding Gli activator gradient

Once in the nucleus, Gli activates gene expression in a concentration-dependent fashion.

The different binding affinity of Gli recognition sequences within the regulatory DNAs of the various target genes is important in the differential regulations of Shh-Gli target genes.

Through what pathways the differential activation of Shh receptors function:

V1 genes can be activated by low levels of Gli because they have high-affinity recognition sequences

In contrast, the V3 target genes might contain regulatory DNA with low-affinity Gli recognition sequences that can be activated only by peak levels of Shh signaling


Cellularization: a process that the zygote transforms to cellular bastoderm

Totipotential: the ability to give rise to any cell type

Part Three: The Molecular Biology of Drosophila EmbryogenesisTerminologies of Drosophila Embryogenesis:

Part Three: The Molecular Biology of Drosophila Embryogenesis

I. InseminationII. “Zygotic” nucleus formationIII. Synchronous divisions and formation

of syncitium-single cell with multiple nuclei

IV. Nuclei migration to cortex and three more divisions

V. Cell membranes formation and loss of totipotential

VI. Transformation into cellular blastoderm

Events of Drosophila Embryogenesis:

A Morphogen Gradient controls Dorsal-Ventral Patterning• The specific morphogen is the Dorsal protein• Dorsal protein enters nuclei in ventral and lateral

regions but remains in the cytoplasm in dorsal regions• Regulated nuclear transport of the Dorsal protein is

controlled by the cell signaling molecule Spätzle, which is distributed in a ventral-to-dorsal gradient within the extracellular matrix

A Morphogen Gradient controls Dorsal-Ventral Patterning

• After fertilization, Spätzle binds to the cell surface Toll receptor. Depending on the concentration of Spätzle, Toll is activated to greater or lesser extent. Peak activation of Toll is in ventral regions, where the Spätzle concentration is highest.

• Toll signaling causes the degration of a cytoplasmic inhibitor Cactus,and the release of Dorsal from the cytoplasm into nuclei.

The twist 5’ regulatory DNA contains two low-affinity Dorsal binding sites

The rhomboid 5’ enhancer contains a cluster of dorsal binding sites but only one has high affinity

The sog intronic enhancer contains four evenly-spaced optimal Dorsal binding sites

A Morphogen Gradient controls Dorsal-Ventral PatterningThree thresholds of gene expression and three types of regulatory DNAs:

Three types of regulatory DNAs:

Both rhomboid and sog gene are kept off by the transcriptional repressor Snail in the mesoderm for they have binding sites for it .Thus the Snail repressor and the affinities of the Dorsal binding sites together determine specific gene expression.


The binding of Dorsal also depends on protein-protein interactions between Dorsal and other regulatory proteins bound to the target enhancers.

For example, intermediate levels of Dorsal are sufficient to bind due to their interactions with another activator protein Twist, they help one another bind to adjacent sites within the rhomboid enhancer.


Segmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Egg

• Two localized mRNAs in the egg at the time of fertilization– The bicoid mRNA - Located at the

anterior pole – The oskar mRNA – Located at the

posterior pole.

The oskar mRNAMovements:

Synthesized within the ovary of mother fly

First deposited at the anterior end of the immature egg by nurse cells

Transported to posterior region when mature egg forms

Functions:Encoding an RNA-

binding protein that is responsible for the assembly of polar granules

The polar granules control the development of tissues that arise from posterior regions of the early embryo

Location of maternal mRNAs

Click here and look into the “adapter” proteins for more details

Location determination The localization of the

bicoid mRNA in anterior regions also depends on sequences contained within its 3’ UTR. Therefore, the 3’UTR is important in determine where each mRNA becomes localized.

If the 3’UTR from the oskar mRNA is replaced with that from biciod, the hybrid oskar mRNA is located to anterior regions (just as biciod normally is).

The Bicoid Gradient Regulates the Expression of Segmentation Genes in a Concentration-Dependent Fashion

The Bicoid regulatory protein I. Synthesized prior to the completion of

cellularizationII. Simply diffuses across the syncitium, which

differs from the case of the Gli and DorsalIII. Produces multiple thresholds of gene

expressionIV. Binds to DNA as a monomer, interacts with

each other to foster the cooperative occupancy of adjacent sites.


High concentrations of Bicoid protein activate the expression of the orthodenticle gene

High and intermediate concentrations of Bicoid are sufficient to activate hunchback


• This differential regulation of orthodenticle and hunchback depends on the binding affinities of Biciod recognition sequences. The orthodenticle gene - 5’ enhancer that contains

a series of low-affinity Biciod binding sites, The hunchback gene - 5’ enhancer contains high-

affinity binding sites.

Hunchback Expression Is also Regulated at the level of Translation

Maternal promoter leads to the synthesis of a hunchback mRNA that is evenly distributed in the unfertilized eggs

The translation of the maternal transcript in the posterior region is blocked by Nanos

Nanos mRNA is located in posterior regions through interactions between its 3’ UTR and the polar granules

Nanos binds to NREs, located in the 3’ UTR of the maternal mRNA, and causes a reduction in the hunchback poly-A tail, which inhibits its translation

Hunchback protein gradient and translation inhibition by Nanos

The Gradient of Hunchback Repressor Establishes Different Limits of Gene Expression

• Hunchback functions as a transcriptional repressor to establish different limits of expression of the gap genes, Krüppel, knirps and giant.

• High levels of the Hunchback protein repress the transcription of Krüppel, whereas intermediate and low levels of the protein repress the expression of the knirps and giant, respectively.

The Gradient of Hunchback Repressor Establishes Different Limits of Gene Expression

Not the binding affinities but the number of Hunchback repressor sites may be more critical for distinct patterns of Krüppel, knirps and giant expression.

Hunchback and Gap proteins produce Segmentation Stripes of Gene Expression

• The eve gene is expressed in a series of seven alternating or pair-rule stripes that extend along the length of the embryo.

• The eve protein coding sequence contains five separate enhancers that together produce the seven different stripes of eve expression


Regulation of eve stripe 2

• Eve stripe 2 contains binding sites for four different regulatory proteins: Bicoid, Hunchback, Giant, and Krüppel.

In principle, Bicoid and Hunchback can activate the stripe 2 enhancer in the entire anterior half of the embryo where they both present

Giant and Krüppel function as repressors that form the anterior and posterior borders, respectively.

Regulation of eve stripe 2


Krüppel mediates transcriptional repression through two distinct mechanisms.– Competition. Two of the three

Krüppel binding sites directly overlap Boicoid activator sites,precludes the activator to bind.

– Quenching. The third Krüppel is able to inhibit the action of the Bicoid activator bound nearby. It depends on the recruitment of the transcriptional repressor CtBP, which contains a enzymatic activity that impairs the function of neighboring activators.

Gap Repressor Gradients Produce many Stripes of Gene Expression

The same basic mechanism of how eve stripe 2 is formed applies to the regulation of the other eve enhancers as well.

The stripe borders are defined by localized gap repressors: Hunchback establishes the anterior border, while Knirps specifies the posterior border.

The differential regulation of the the two enhancers by the repressor gradient produces distinct anterior borders for the eve stripes.

The eve stripe 3 enhancer is repressed by high levels of the Hunchback gradient but low levels of the Knirps gradient

The stripe 4 enhancer is just the opposite, this differences are due to the number of repressor binding sites.

Gap Repressor Gradients Produce many Stripes of Gene Expression

Short-range Transcriptional Repressors Permit Different Enhancers to Work Independently

There are additional enhancers that control eve expression,this type of complex regulation is common.

The mechanism that repressors bound to one enhancer do not interfere with activators in the neighboring enhancers is short-range transcriptional repression,which ensures enhancer autonomy.

Stripe 3 activator is not repressed by the Krüppel repressors bound to the stripe 2 enhancer because it lacks the specific DNA sequences that are recognizes by the Krüppel protein and they map too far away.

Short-range Transcriptional Repressors Permit Different Enhancers to Work Independently

Brief Summary:

I. There are three major ways to regulate gene expression at the level of transcription initiation

II. The segmentation of the Drosophila embryo depends on a combination of localized mRNAs and gradients of regulatory factors

May God be with you!

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Gene Regulation during Development 200431060022 陈国荣